Antibiotics and their effects on Plants

Soil bacteria and fungi are a rich source of natural antibiotics, but the prevalence of human-made antibiotics and antibiotic resistance genes in soils, is an emerging concern. Antibiotics are widely used to promote livestock growth in modern non-organic agriculture, with poultry, cattle and pigs, being regularly treated with these antibacterial drugs. Millions of kilograms of antibiotics are released into the environment annually, much in the excrement of grazing animals, or through application of manure to agricultural fields(1). Discharge of human waste into waterways and the use of contaminated irrigation water or sewerage sludge to fertilise crops in many countries, is also a contributory cause. As a result, a higher level of antibiotic resistance is now apparent in conventional agricultural versus natural forest soils(2).

Soil and water-containing antibiotics constitute a potential route of human exposure to antibiotic resistance genes through their uptake by plants(3-8).  Uptake by plants can also have other effects, such as the accumulation of nitrofuran-type antibiotics in the edible parts of spring onions, and the subsequent metabolism of these into genotoxic and potentially carcinogenic hydrazine-containing metabolites(9).

The other consideration is the effects these human-made antibiotics have on the soil or plants themselves.  With human and animal health being intrinsically connected to that of plants and soil, and increasing research showing the many symbiotic and complex relationships between living organisms and their environment, effects of human-made antibiotics on plant health, should also be considered.

The high level of contamination with antibiotic residues and transferable resistance genes in pig manure applied to soil, has been shown to change the antibiotic resistant gene reservoir of the plant microbiome(10).  Carrots and lettuce can uptake amoxicillin and tetracycline(4), and tetracycline residues have toxic effects on both root and stems of germinating lettuce seedlings(11).  Oxytetracycline residues from cattle manure have also been shown to affect the diversity and type of nitrogen-fixing soil bacteria communities(12).

A recent European study has shown that even small amounts of antibiotics can have a range of potentially negative effects on plant traits(13). The comprehensive study examined the effects of three antibiotics (penicillin, tetracycline and sulfadiazine), on germination and growth of four plant species. These included two cultivated species (rapeseed, Brassica napus and common wheat, Tricicum aestivum), and two non-crop (herb) species (Shepherd’s purse, Capsella bursa-pastoria and Common Windgrass, Apera spicaventi). In farmland fertilised with manure containing antibiotic concentrations as typically found in agricultural soils, various effects on the plants were observed.

Main effects were delayed germination or reduced plant biomass. These effects varied markedly depending on the plant species concerned, but were most pronounced in the two herb species, particularly by penicillin and sulfadiazine. This suggests that different antibiotics could potentially affect the prevalence and types of species, and the diversity of natural plant communities near agricultural fields. Furthermore, these species-specific responses may not only alter the competitive abilities and makeup of the plant community, but also have secondary effects on other species such as pollinating and herbivorous insects(13).

Petrochemical residues and the use of non-organic agricultural pesticides and insecticides, are also starting to come under the spotlight as likely contributors to multi-drug antibiotic resistance among soil bacteria. A recent Chinese study has demonstrated that petrochemical residue -polluted soils were more than 15 times more likely than less-contaminated ones, to contain antibiotic resistance genes. This strong association of soil pollution with polycyclic aromatic hydrocarbons, suggests these may also be contributing to the growing amounts of antibiotic resistant genes in human-impacted environments(14).

In non-organic agriculture, soil bacteria can be continuously exposed to synthetic pesticides at sub-lethal concentrations, and a recent Indian study has found that insecticide-contaminated soil may have contributed to development of resistance to a range of different antibiotics, by several Bacillus species(15).

Silver nanoparticles are also now widely used in antibacterial products, and these inevitably discharge into aquatic environments and have been shown to affect the nitrogen cycle in phytoplankton and aquatic plant life(16).

Antimicrobial chemicals such as triclosan and triclocarban, which are used in some liquid soaps and toothpastes, can take a long time to break down in the environment and have been shown to have detrimental effects on aquatic organisms, and potentially contribute to antimicrobial resistance(17-19).

Soil and plant health are pivotal to the health of the planet and all its living organisms, and antibiotic drugs have saved many millions of lives. However, the widespread use of antibiotics in non-organic agricultural production systems particularly those involving animals, should be curtailed.

Refs:

  1. Popova IE et al, J Environ Sci Health B 2017; 52(5):298-305.
  2. Popowska M et al, Antimicrob Agents Chemother 2012; 56(3):1434-1443.
  3. Grote M. et al, Landbauforschung Volkenrode 2007; 57: 25-32.
  4. Azanu D et al, Chemosphere 2016; 157:107-114.
  5. Rahube TO et al, Can J Microbiol 2016; 62(7):600-7.
  6. Pan M et al, J Agric Food Chem 2014; 62:11062-11069.
  7. Kang DH et al, J AGric Food Chem 2013; 61:9992-10001.
  8. Kumar K et al, J Environment Qual 2005; 32:2082-2085.
  9. Wang Y et al, J Agric Food Chem 2017; 65(21):4255-4261.
  10. Wolters B et al, Appl Microbiol Biotechnol 2016; 100(21):9343-9353.
  11. Pino MR et al, Environ Sci Pollut Res Int 2016; 23(22):22530-22541.
  12. Sun J et al, Bioresour Technol 2016; 801-807, epub May 21.
  13. Minden V et al, AoB Plants 2017; 9(2):plx020.
  14. Chen B et al, Environ Pollut 2017; 220(Pt B):1005-1013.
  15. Rangasamy K et al, Microb Pathog 2017; 103:153-165.
  16. Jiang HS et al, Environ Pollut 2017; 223:395-402.
  17. Falisse E et al, Aquat Toxicol 2017; 189:97-107.
  18. McNamara PJ, Levy SB. Antimicrob Agents Chemother 2016; 60(12):7015-7016.
  19. Tremblay Louis, Environmental toxicologist, Cawthron Institute, Nelson, New Zealand Herald, 23 June 2017.

Manuka and Myrtle Rust

Last week I attended a two day workshop organised by scientists at Plant and Food Research Ltd and Massey University in Palmerston North, to discuss a range of recent scientific and biosecurity developments, concerning Manuka (Leptospermum scoparium), an important plant in New Zealand’s natural environment and economy. As with the two day Hui on ‘Manuka and More’ in Ruatoria and Te Araroa in November last year, this was an excellent event in which more than 30 scientists working actively on Manuka research presented on a diverse range of subjects and discussed where there could be gaps in our knowledge or research needs for this plant. While Manuka Honey and essential oil are currently the main two medicinal products produced from Manuka, numerous other therapeutic applications and potential contributions to preserving our environment, are found within this plant.

Jacqui Horswell and colleagues from the Institute of Environmental Science and Research, have shown that Manuka and other myrtaceaeous plants seem to be capable of killing the faecal bacterial pathogen Enterobacter coli (E. coli), by enhancing the die-off of this and other pathogenic organisms that pass through their root systems. A field trial involving riparian planting of Manuka is just getting going, to see whether laboratory results extend to helping to reduce animal effluent flows into a polluted lake. A lake which was once pristine and a treasured swimming area, but in recent years has changed into a green and dirty waterway due largely to dairy industry runoff, has been selected for this trial.

Hayley Ridgway from Lincoln University presented some interesting findings concerning novel and potentially useful mycorrhizae (fungi) and endophytic bacteria associated with the roots of Manuka, some of which I wrote about in my previous blog. Inoculation of Manuka plants with different mycorrhizae causes significant alterations in their growth rates and essential oil composition, highlighting the complex inter-relationships between microbes associated with Manuka, and its production of phytochemicals including some with bioactive properties.

Other presentations were made on experiences to date involving plantations of Manuka which have been established at a number of North Island sites in recent years. Challenges include site access, weeds, pests, and the relative attractiveness of different genetic lines to bees. A comment made by one of the presenters that while humans have had multiple generations of experience with cultivation and enhancing performance characteristics of crops such as wheat and rice, our experience with Manuka plantations spans less than 10-15 years to date.

The hottest topic at the workshop, however, was the recent finding of isolated outbreaks of Myrtle Rust (Austropuccinia psidii) in New Zealand nursery and garden grown specimens of Manuka and the native tree, Ramarama (Lophomyrtus bullata). This pathogenic fungi originated from Brazil where it causes guava rust, but spread internationally into North America in the 1880’s, and was first reported in Australia in 2010.  Australia is home to around half of the world’s Myrtaceae (Myrtle family) plant species, including Eucalyptus (850 species), Melaleuca (176 species) and Callistemon species.

Outbreak of Myrtle rust has had a devastating effect on much of the east coast as well as other areas of Australia, where it has resulted in ecosystem collapse for certain plant species. To date it has only been found in isolated locations in Northland, Waikato, Bay of Plenty and Taranaki, although it is widespread on Raoul Island in the Kermadec group, about 1,100km to the north-east of New Zealand.

Myrtle rust spores can easily spread across large distances by wind, or via insects, birds, people, or machinery, and it is thought the fungus arrived in New Zealand carried by strong winds and significant weather events from Australia.

The Myrtle Rust Strategic Science Advisory Group is working hard to assess and try to ameliorate the widespread environmental, economic, social and cultural impacts this plant pathogen could have on New Zealand. Apart from Manuka and Ramarama, other indigenous Myrtaceae species such as Pohutakawa (Metrosideros spp) and Swamp Maire (Syzygium maire), are under risk. Priorities including acceleration of scientific research into the biology of the pandemic strain detected here, pathways of spread, surveillance, management, exploring plant susceptibility and resistance, and coordinating and communicating a management plan that has widespread engagement by communities, scientists, industry and Maori stakeholders and landowners, councils and government.

The Ministry for Primary Industries (MPI) and the Department of Conservation (DOC), with the help of local iwi, the nursery industry, and local authorities are running an operation to determine the scale of the situation and to try and contain and control myrtle rust in the areas it has been found. However, emergence of the infection and appearance of the distinctive yellow or brown leaf discolouration may not become fully apparent until the spring, and a better assessment of the number of infection sites and their extent, may not be possible until then.

The arrival of Myrtle Rust in New Zealand means that the task of collecting and storing seed of New Zealand indigenous Myrtaceae including Manuka, has now become urgent. The NZ Indigenous Flora Seed Bank (NZIFSB), a collaborative project between Massey University, AgResearch, Landcare and the Department of Conservation, with support from the NZ Plant Conservation Network and the Millennium Seedbank at Kew in the UK, was established in 2013. NZFISB has been doing some really valuable work to collect and store seeds aimed at preserving a wide range of biodiversity within New Zealand native plant species. More than 130 volunteer seed collectors have been trained to date, and plans are underway to extend this and the level of community participation, to try to better protect our native plants for generations to come.

Refs:

http://www.nzpcn.org.nz/page.aspx?conservation_seedbank

http://www.mpi.govt.nz/protection-and-response/responding/alerts/myrtle-rust/

Antimicrobial Endophytes in Echinacea, Olive and Manuka

While plants are being extensively explored for new therapeutic properties and pharmacological activities, the communities of live fungi and bacteria known as endophytes that live between living plant cells, are also now being regarded as having many useful potential medicinal applications. Ironically, in recent years it is these microorganisms associated with plants rather than plants themselves, which seem to be receive much research interest.

Endophytes are microorganisms that live within a plant for at least part of their life cycles, without causing apparent disease or infections in the plant. Different endophytes seem to have affinities for particular plants, with which they have distinctive and cherished but complex interactions while each of them grows. They are for instance known to sometimes enhance host growth and nutrient gain, improve the plant’s ability to tolerate various types of stressors, and enhance the its resistance to insects and pests. The rrelationships that these bacteria and fungal communities have with their host plant varies from symbiotic to parasitic, to bordering on pathogenic.
Some very unusual and valuable bioactive substances are sometimes produced by these endophytes, such as alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids, and these are increasingly being recognized as sources of novel compounds which may help to maintain or solve not only the plant’s health challenges, but can also have applications in human and animal health problems.
Over the past few decades, some highly medicinal compounds produced by endophytic microbes lead to novel drug development. These include Taxol (paclitaxol), a complex diterpene alkaloid produced by the endophyte Metarhizium anisopliae found in the bark of the Pacific Yew (Taxus brevifolia) tree, and one of the most promising anticancer agents ever developed. Also streptomycin, an antibiotic produced from the bacterial endophyte Streptomyces.

Other endophytes possess antibacterial activities which may be useful in treating various infections, and in a world where antibiotic resistance is becoming a major public health threat, these are obviously of great interest. Exploring and bioprospecting these for potential antimicrobial compounds may well yield valuable new natural products or drugs to help in the fight against resistant organisms(1,2,3,4).

It now seems that bacterial communities colonizing Echinacea purpurea contribute to its well-known immune enhancing activity(5). American researchers have reported that Echinacea’s stimulating activity on monocytes (a type of white blood cell involved in engulfing and destroying harmful microbes), could be solely if not partially accounted for by the activities and prevalence of Proteobacteria, a family of bacteria found in the bacterial community associated with this medicinal plant.
A screen of 151 different endophytic bacteria isolated from three different compartments of Echinacea purpurea, revealed that several bacteria isolated from the roots are strong inhibitors of Burkholderia cepacia complex bacteria, a serious threat particularly in immune-compromised cystic fibrosis patients(6). One of these bacterial strains also showed antimicrobial effects against Acinetobacter baumannii, a pathogenic bacteria mainly associated with hospital-acquired infections, and Klebsiella pneumoniae, also increasingly incriminated in hospital infections(7). Interestingly, the type of bacteria and their antimicrobial effects varied considerably, according to which part of the plant (root, stem, leaves etc) they were associated with. This has resemblances to different plant parts of Echinacea having different phytochemical and thus pharmacological activities, such as Echinacea roots being richest in alkylamides and thus anti-inflammatory activities.

Endophytic fungi including Penicillium commune and Penicillium canescens (related to the Penicillium notatum mould from which the first antibiotic penicillin originated), have also been isolated from the leaves of olive (Olea europaea) trees, and several of these have also shown antibacterial as well as antifungal activities in recent work(8).

Finally, a rich endophyte community has recently been identified by Lincoln University researchers for the New Zealand native plant Manuka (Leptospermum scoparium). A total of 192 culturable bacteria were recovered from leaves, stems and roots, including some showing activity against the bacterial pathogen, Pseudomonas syringae pv. actinidiae(9), otherwise known by Kiwifruit growers as Psa. With Psa being a serious risk to the health of the Kiwifruit vine, it could be that these endophytic bacteria found within Manuka will make a useful contribution to ensuring the future health of the Kiwifruit industry.
While very few of all of the world’s plants have had their complete complement of endophytes studied, these are just three well established medicinal plants from which some highly active cohabitating bacteria and fungi have been sourced. Undoubtedly this area of research will receive much more attention due to growing concerns about antibiotic resistance, as there would seem to be a huge opportunity to find new and interesting endophytes among the wealth of different plants growing not only in soil, but also in waterways and oceans.
Refs:
1. Alvin A et al, Microbiol Res 2014; 169(7-8)L483-495.
2. Martinez-Klimova E et al, Biochem Pharmacol 2016; Oct 27.
3. Kealey C et al, Biotechnol Lett 2017; Mar 8 (epub ahead of print)
4. Tanwar A et al, Microbiol Path 2016;101:76-82
5. Haron MH et al, Planta Med 2016; 82(14):1258-1265.
6. Chiellini C et al, Microbiol Res 2017; 196:34-43.
7. Presta L et al, Res Microbiol 2017; 168(3):293-305.
8. Malhadas C et al, World J Microbiol Biotechnol 2017; 33(3):46.
9. Wicaksono WA et al, PLoS One 2016; 11(9):e0163717.

Calendula for venous ulcers and thrush

Preparations made from flowers of the English or Pot Marigold (Calendula officinalis), a well-known garden plant, are widely recommended and used by western medical herbalists for minor cuts, grazes, and slow healing wounds, where an antiseptic and healing action is required. The European Medicines Agency has also approved Calendula as a traditional medicinal product for the treatment of minor skin inflammation, and as an aid in the healing of minor wounds.

However, apart from two clinical trials showing benefits from a Calendula ointment to treat dermatitis secondary to radiotherapy treatment in cancer patients (1, 2), few human clinical trials have been published.

Brazilian researchers recently conducted a clinical trial to evaluate the effectiveness of Calendula extract in the treatment of non-healing venous leg ulcers (3). Treatment consisted of a twice daily spray application of a hydro glycolic extract of Calendula, or the standard hospital procedure which included the use of collagenase, the antibiotic chloramphenicol, and 1% silver sulfadiazine cream. Both treatments were given for a period of 30 weeks or until ulcers healed, and nursing staff examined and assessed patients every two weeks, during which the wound area was measured and clinically evaluated.

Swabs were collected from the wounds before starting treatment, and these showed Staphylococcus aureus to be the predominant microorganism in both the control and Calendula groups, with Pseudomonas spp., Klebsiella spp., and Escherichia coli being found in the wounds of some patients. The size and duration of ulcers prior to treatment was similar in both groups, and patients had an average age of 68 years in the control group of 19 patients, and 63 in the Calendula treated group of 38 patients.

After 12 weeks treatment, 39% of Calendula-treated wounds were completely closed, but none in the standard treatment control group. After 30 weeks treatment, 72% achieved complete wound closure, while only 32% were closed in the control group. An average of 12 weeks was needed for healing to occur in the Calendula-treated group, but 25 weeks in the control group. No adverse events were observed during the Calendula treatment.

Favourable outcomes were also achieved in a Serbian clinical trial which involved 21 venous ulcer patients who applied Calendula ointment twice daily for 3 weeks (4). Calendula treatment reduced the total surface of the ulcers by an average of 41.7%, but only by 14.5% in the control group of 13 patients treated with saline dressings. Complete wound closure was achieved in four patients treated with Calendula.

These two studies show a significant acceleration of chronic venous ulcer wound healing through twice daily application of topical products made using Calendula flowers. While a 3 week treatment period was beneficial, continuing this for longer periods is recommended to achieve optimal results.

Beneficial effects of Calendula cream for the treatment of vaginal Candidiasis (thrush), were also reported from a recent Iranian study involving 150 women aged 18-45 years(5). Participants used a 5-g vaginal applicator of Calendula or clotrimazole (an antifungal drug) cream for seven nights before going to bed, then were assessed at two post treatment time points.

Compared to the clotrimazole group, fewer women in the Calendula group tested negative for Candidiasis at 10–15 days following treatment (49% vs 74%), although both treatments had similar effects at improving clinical signs and symptoms and sexual function. At a 30-35 day post treatment follow up, however, signs and symptoms were less severe in the Calendula treated group, and women treated with Calendula were significantly less likely to have suffered a recurrence of Candidiasis, than those treated with clotrimazole.

These results suggest that a seven day course of intravaginal calendula cream is safe and can be effective for the treatment of vaginal Candidiasis, but seems to have a delayed effect compared to clotrimazole. However, Calendula use may have better long term outcomes such as a lower risk of recurring infections, even after a 7 day course.

While a longer duration of Calendula use may have greater effectiveness in the treatment of vaginal Candidiasis, particularly as resistance is increasing to clotrimazole and other antifungal drugs, this trial provides encouraging data to support its use for this annoying problem experienced by a large number of women.

Clearly the impressive healing capabilities held by the attractive and bee-loving flowers of this easy to grow plant, extend beyond minor cuts and grazes, to include more chronic and debilitating conditions such as venous ulcers and vaginal thrush.

Refs:

  1. Pommier P et al, J Clin Oncol 2004; 22(8):1447-1453.
  2. Schneider F et al, Rev Esc Enferm USP 2015; 49(2):221-228
  3. Buzzi M et al, J Wound Care 2016; 25(12):732-739
  4. Duran V et al, Int J Tissue React 2005
  5. Saffari E et al, Women & Health 2016; Nov 23:1-16

 

Herbal Medicine Can Help Reduce High Demands on Hospitals

An article in yesterday’s New Zealand Herald , reports that Auckland City Hospital is struggling to cope with a huge increase in demand which has seen almost 200 people coming through the emergency department every day. This increase in patients (7% up on those treated at the same time last year) has also been experienced by a large number of hospitals throughout the country, at a time of year when the impact of seasonal illnesses such as influenza, has yet to manifest.

While New Zealand’s population growth is a contributory factor, A&E departments are seeing a higher proportion of sicker and older people than in the past, as well as chronic illnesses such as diabetes, heart disease and cancer that were less common in days gone by. Additionally, the impact of prolonged poverty and poor diets in too high a proportion of NZ’s population, continues to be avoidable catalysts to a need for emergency hospital services.

The fact that Auckland Hospital’s capacity has been in the high 90’s and has hit 100% a couple of times recently, combined with more and more seriously sick patients requiring treatment, is cause for alarm. Should an influenza epidemic or major disaster occur during the next few months, the ability for existing healthcare services to cope with the sudden increase in additional demand, is in serious doubt. Given the huge and growing pressures they are under, many nurses, doctors, and other healthcare professionals are already close to breaking point, and it is therefore, essential that we reflect upon how this unacceptable situation can be addressed.

Herbal Medicine (Phytotherapy) is the oldest and most used form of medicine in the world. While like most people I am extremely grateful to be living in an age where drugs such as antibiotics and modern medical interventions can treat conditions that once would have resulted in an early death, it is time for the potential contribution of plant-based medicine to human wellness and many common illnesses, to be better recognised.

Evidence for the therapeutic benefits of many herbs and plants incorporated into the diet or an overall treatment approach has increased exponentially in recent years, and to not take this evidence seriously, will be to our peril. A vast body of science suggests that a phenomenal number of plants can either enhance our resistance to and reduce our risks of a wide range of illnesses, or reduce our over-dependence on drug and hospital treatments.

While a large number and diverse array of herbal products are easily obtained from retail outlets or online from e-commerce sites, and many of these are appropriate to take for minor ailments or to help prevent illnesses such as headaches, stomach upsets, colds and the flu, other herbs and a more systematic treatment for more serious or chronic conditions, are best accessed through consulting a properly trained medical herbalist. Such practitioners in New Zealand generally spend 3 or 4 years in full-time training within accredited institutions to study physiology, anatomy, biochemistry, diagnosis and nutrition, and how to prescribe herbal medicines and avoid adverse herb-drug interactions, and most are professional members of the New Zealand Association of Medical Herbalists (NZAMH) . In the same way that we go to a cardiologist for a comprehensive check-up and full diagnosis before being prescribed medications for a heart condition, the professional medical herbalist should be regarded as the specialist we consult before taking herbs for any serious, chronic or debilitating illness.

Apart from their skills in prescribing effective herbal medicines, medical herbalists and naturopaths can make a significant contribution to encouraging healthier diets, lifestyles and other activities that could have a large impact on reducing the number of hospital admissions and pressure on A&E services.

Last week I gave a presentation to a Brisbane conference organised by the Naturopathic and Herbalists’ Association of Australia (NHAA), on the subject of Ginkgo, Ginger and Ginseng, and their use as adjunctive herbs alongside conventional medicine. These are just 3 herbs for which there is compelling evidence that taking them together with various drugs for conditions including dementia, schizophrenia, infectious disease and diabetes, is not only more effective, but can also reduce drug-related adverse effects, and have pharmacoeconomic benefits through reducing the dependence on costly conventional healthcare services. These are just the tip of the iceberg in what else we can achieve through proper and professionally supervised combinations of herbs with drugs, and more research funding and clinical trials are warranted.

As with all health professionals, however, regulation by government to ensure that an adequate standard of service is provided and that the practitioner works in a professional and ethical manner with his or her patient, is essential. The NZAMH is a body that recognised this need a long time ago, and first applied in 2007 for statutory regulation under the Health Practitioners’ Competence Assurance (HPCA) Act (legislation which regulates a number of different health practitioner professions). While this application was subsequently accepted by the then Minister of Health, Pete Hodgson, with a change in government and revision of the HPCA Act soon afterwards, NZAMH was told a new application was needed. A resubmitted application was made to the government in early 2015, but disappointingly, there seems to have been little progression of it since that time.

During World War 2, when New Zealand’s access to many drugs and conventional medicines was under threat, much ground-breaking and valuable research was undertaken into the phytochemistry and bioactivities of many of our indigenous flora and fauna. With our hospitals now close to bursting, not to mention an alarming global increase in antibiotic resistance, the time has now come for policy makers, health funding providers, other health professionals and politicians, to again cast their attention to the enormous contribution that herbal medicine can make to our current and future healthcare services.

Why Herbs Should Be the First Choice of Treatment for Acute Anxiety

Anxiety can manifest in a wide range of ways. Apart from the internal emotional fearfulness, symptoms can include irritability, agitation, muscle tension, palpitations, sweating, insomnia, breathlessness, poor concentration, reduced socialisation and ability to undertake everyday activities. It is the most prevalent mental health disorder affecting children and adults, but many more people are dealing with problematic anxiety symptoms without any diagnosis.

In our increasingly changing world, where our daily exposure to stressful stimuli and life challenges can produce a rising barometer of worries, anxiety is often a major impediment to leading a fulfilling and happy life. Like most other health woes, humans have long pursued various practices to help overcome anxiety, the most popular of which is alcohol. Then there are drug medications, which have long been used to relieve anxiety, and remain widely prescribed.

Barbiturates were the first of these, sedative and anticonvulsant drugs which became popular particularly with sleep-deprived young mothers in the middle of last century, but which lead to the overdose deaths of thousands of people, including Elvis Presley and Marilyn Monroe. The next day ‘hangover effect’ from barbiturates was also always a problem, and development of a new chemical group of anxiolytic (anti-anxiety) and sedative drugs known as the benzodiazepines, lead to these superceding the barbiturates for the treatment of anxiety and insomnia. Benzodiazepines seem to act predominantly through stimulating GABA (gamma amino butyric acid) receptors in the central nervous system, and the commercialisation of Valium® (diazepam) by Roche in 1963 marked the start of a period during which this and other benzodiazepine drugs such as lorazepam, alprazolam and clonazepam began to be widely prescribed by GP’s and psychiatrists for anxiety and sleep difficulties. Between 1969 and 1982 Valium® was the most prescribed drug in the U.S., during which time Roche’s share price soared.

While safer than barbiturates, and effective as a ‘quick fix’ for anxious feelings or insomnia, safety concerns for benzodiazepines soon emerged. Feelings of fatigue, or a hangover the following day when taken as sleeping tablets, and a wide range of other side effects are all too common experiences. Most significant of these is the development of tolerance when they are used for more than a short period of time. As anyone who has been through it will testify, withdrawing from long term benzodiazepine use is a hugely stressful, unpleasant and often very protracted experience.

Feelings of depression can both contribute to or arise from excessive anxiety, and it is not uncommon for feelings of low mood and a low tolerance to stress, to be experienced together with anxiety. Apart from GABA, neurotransmitters such as serotonin, adrenaline and dopamine are intrinsically involved in influencing our emotions and mood, interacting together in complex ways that scientists still have little understanding of. It is therefore not surprising that many SSRI’s (selective serotonin reuptake inhibitor) drugs, used primarily as antidepressants, can have an anxiolytic effect in some people, and in many countries, these are often prescribed instead of or together with benzodiazepines, for anxiety conditions.

While sometimes effective as anxiolytics and less likely to produce adverse effects than most older generation tricyclic antidepressants, some find that SSRI’s can cause or increase anxiety feelings, or experience any one or more of a wide range of unpleasant side effects including insomnia, weight gain, emotional numbing or sexual dysfunction.

Another class of non-benzodiazepine sleeping tablets, the so-called  ‘Z-drugs’ such as zopiclone and zolpidem, have become popular in recent years, and while initially thought to be less habit-forming than benzodiazepines, they can also be very difficult to withdraw from after more than short-term use.

A large number of herbs have been traditionally used for nervous conditions and their anxiolytic effects, several of which have been shown in clinical trials to be beneficial as anxiety treatments. These include Chamomile, Skullcap, Passionflower, Valerian, Kava, Lemon balm and Withania.  Despite the number of well-designed trials undertaken to date being relatively low, and results sometimes variable depending on the particular herbal product(s) and dosages used, results are encouraging and in all cases show a better safety profile than for comparable anxiolytic drugs.

Of these, Kava (Piper methysticum), is the most studied, and is a non-addictive anxiolytic with great potential to treat anxiety. Its effectiveness in treating anxiety has been affirmed through several clinical trials and meta-analysis(1-3). While case reports of liver toxicity associated with kava usage lead to its restriction in some countries at the end of last century, use of the wrong plant part as raw material, or use in combination with alcohol or various drugs, were likely contributory factors. Also the frequency of such adverse events reports was substantially less than that for paracetamol, a commonly used analgesic.

Aerial parts of the herb Passionflower (Passiflora incarnata), have also been taken for anxiety for many centuries, and in a trial involving 36 outpatients with generalized anxiety disorder, it was as effective as the benzodiazepine drug oxazepam, but unlike oxazepam caused no impairment of job performance(4).

Roots of the herb Withania (Withania somnifera, Ashwagandha), have a subtle but powerful nervous system and adrenal tonic action, which insulates the nervous system from stress, and enables the adrenal glands to be better prepared to respond appropriately to stressful stimuli. A large number of scientific papers now support its applications for stress-associated anxiety conditions, including several recent human clinical trials(5,6).

While further studies involving greater participant numbers and longer term treatment are needed to identify optimal dosages and phytochemical makeup of the treatments involved, the fact that most herbal anxiolytic agents are safe and have the same or only a slightly higher incidence of adverse effects to placebo, is clear. It is therefore logical that before reverting to drug medications, more likely to produce unwanted adverse effects and in some cases long term dependency, herbal anxiolytics should be tried, in anxiety conditions.

Refs:

  1. Sarris J, Aust NZ J Psychiatry 2011; 45(1):27-35.
  2. Sarris J, J Clin Psychopharmacol 2013; 33(5):643-648.
  3. Savage K et al, Trials 2015; 16:493.
  4. Akhondzadeh S et al, J Clin Pharm Ther 2001; 26(5):363-367.
  5. Chandrasekhar K et al, Indian J Psychol Med 2012; 34(3):255-262.
  6. Pratte MA et al, J Altern Complement Med 2014; 20(12):901-908

Manuka & More

I recently attended a very interesting Hui (Gathering) in Ruatoria and Te Araroa on New Zealand’s East Coast, entitled ‘Manuka and More’.  Around 15 researchers from Crown Research Institutes and industry representatives including myself gave talks on subjects related to the NZ native tree Manuka (Leptospermum scoparium), which grows prolifically around the coast, and provides nectar for honeybees which produce manuka honey.  Manuka honey is being increasingly recognised as a highly active natural product with benefits as an antimicrobial and wound healer, and global demand for it has soared in recent years. Similarly the volatile oil of manuka has antimicrobial and anti-inflammatory properties, and is increasingly sought after.

manuka-4
Studies into what makes manuka honey so special, and characterisation of its many different chemotypes and genotypes, has been a focus of much research in the past decade. To the East Coast locals, manuka was once regarded mainly as a scrub plant and nuisance that was cleared to make way for pastural farming of sheep and cattle, but with honey prices continuing to rise and there being little money now in wool, manuka is being allowed to re-establish itself in many areas. Additionally, a lot of effort is now going into planting nursery-raised seedlings bred from chemotypes thought to produce optimal quality and yields of honey and oil.

With the plantation model being in its relative infancy, research into the potential effects of planted manuka on the local pre-existing chemotypes, and whether the yield of honey or oil will in fact be as high as hoped from these cultivated plants, is an area for ongoing investigation.

A growing number of local East coast people and Maori-controlled enterprises are now getting into the honey producing business, and the number of hives in NZ has nearly doubled from around 350,000 to 700,000 over the past 5 years. The sustainability of this level of honey production is another area requiring research, particularly as bees only feed off manuka (and kanuka) nectar for around 6 weeks each season. Monitoring their activities and ensuring they have sufficient food for the remaining 46 weeks of the year, is important.

Of the various flowering plants NZ honey bees feed off, Willow trees (Salix species), are an important source of pollen and protein for bees to feed their brood in the spring time, thus helping them to expand their population and gain maximum strength before the start of the honey flow season. Around the East coast a large number of willows grow particularly along waterways and on erosion prone areas. While the biggest problems for young willows are grazing animals and pests such as possums, rabbits and hares, an emerging pest is also the giant willow aphid which first appeared in NZ in 2013. Apart from infesting willow trees, this can boost the populations of wasps that attack honey bees.

smaller-leptospermum-scoparium-flower-manuka-flower-julyWhile not pleasing to all, other flowering plants such as the invasive introduced gorse (Ulex europaeus), presently plays an important role as a food source for bees in some areas. However, we should be planting other native species such as Hoheria (Hoheria populnea),  Whauwhaupaku or Five Finger (Pseudopanax arboreus) and many others, to provide pollen and nectar as a replacement for that from this imported thorny plant.

Other research presented at the Hui related to the role that mycorrhizal fungi, which grow on the roots of most plants, may have in ensuring the health of the manuka shrub. Most plants co-exist with these fungi, which help them better absorb nutrients from the surrounding soil, and can also help with disease prevention. Also monitoring for potential disease or infestation threats to Manuka such as Myrtle rust, a serious fungal disease not present in New Zealand, but which can affect other plants in the myrtle (Myrtaceae) family.

Recent studies suggesting that manuka seems to be useful at soaking up excremental pollution, and thus may be an ideal tree to plant alongside waterways polluted by effluent runoff from our overly intensive dairy industry, point to yet another exciting development in our understanding about this amazing native plant.

Overall, the range and quality of the diverse areas of research being undertaken, was most encouraging. This combined with the hands-on experience and traditional knowledge of the local Ngati Porou people who are increasingly finding meaningful employment opportunities from manuka-based businesses, gives great encouragement to the future social, economic and environmental wellbeing, of this beautiful area of New Zealand.

Manuka Oil as an Alternative to Antibiotic Creams

New Zealand has a higher incidence of Staphylococcus aureus infections than anywhere else in the developed world, and there has been a significant increase in the number of infections over the past decade, with Māori and Pacific children particularly affected.

manuka-flower-macro

Over usage of an antibiotic is likely to have contributed to this paradoxical increase in serious skin infections, according to results from a Health Research Council funded study. This revealed an increase in the prevalence of resistance in Staphylococcus aureus from 17% in 1999, to 28% in 2013. Dr Deborah Williamson, the clinical microbiologist who lead the study, made the statement in a recent press release that “The increase that we’ve seen in the incidence of serious skin infections in New Zealand children has happened at the same time as an increase in the dispensing of topical fusidic acid to treat skin infections”(1).

Fusidic acid is an antibiotic derived from the fungus Fusidium coccineum and was first released for clinical use in the 1960’s. A 2% fusidic acid cream is currently recommended as a first-line treatment for serious skin infections such as impetigo (school sores), infection of the hair follicles and boils. Most of these are due to the bacteria Staphylococcus aureus, including the notorious methicillin resistant Staphylococcus aureus (MRSA). Like all antibiotics, drug resistance can develop, and this is invariably at a rate proportionate to the extent of usage.

In a paper published in the New Zealand Medical Journal last December, Dr Williamson reviewed the history and usage of topical antimicrobials in New Zealand (2).

This painted a somewhat alarming picture, and the fusidic acid story is an all too familiar one. Another topical antimicrobial agent widely used throughout the 1990s, mupirocin (Bactroban©), was for many years made available to purchase ‘over-the-counter’ (OTC). This led to high levels of use, and subsequent high rates of resistance, and by 2000, approximately 14% of S. aureus isolates displayed high-level resistance to mupirocin(3) . From April, 2000, regulatory changes lead to mupirocin being restricted again to ‘prescription only’, and the resulting decreased usage lead to a fall in the prevalence of high-level mupirocin resistance in S. aureus from 14.2% in 2000, to 8.3% in 2014 (4).

New Zealand is not alone in having a high rate of bacterial resistance to topical antimicrobials, and resistance to antibiotics poses a major global threat, according to a 2014 report by the World Health Organisation(5). Resistance is happening in every region of the world, and unless some major developments take place soon, humankind could be heading towards a time when once again, antibiotics cannot be relied upon to protect against simple infections including those that are risk factors associated with surgery. Development of strategies to mitigate further increases in antimicrobial resistance to topical treatments, is urgently required(6, 7).

Key to this, should be effective wound management. This should combine mechanical-chemical procedures such as debridement, antiseptics, and antimicrobial supportive compresses to help remove the biofilm (an association of microbes and slime which adheres to the surface of the wound, delaying granulation tissue formation and migration of epithelial cells).

Limitation of the level of usage of drug-based antimicrobials, or using two or more of them together rather than alone, and avoidance of topical antibiotic use in common conditions such as acne, are other ways to help reduce the likelihood of resistance(8).

Plants contain a large number of diverse chemicals (phytochemicals) which they produce as defence tools to enable them to survive in their particular environment, and some of these have potent antibacterial activities which can help us fight a wide range of common skin infections.

manuka-2The New Zealand native Manuka (Leptospermum scoparium) is one of these, and the ability of certain forms of Manuka Honey to act as potent healing agents for wounds and ulcers, is becoming increasingly recognised(8). Many clinical trials have now shown manuka honey dressings to have unique healing properties in chronic leg ulcers and other stubborn skin infections, and synergistic antimicrobial activities with various antibiotics, have recently been reported(10,11).

Manuka’s medicinal properties extend way beyond those of the honey that bees manufacture from its pollen, however, and other parts and extracts of this wonderful plant, have therapeutic activities. Manuka essential oil has also been shown to exhibit powerful antimicrobial properties, particularly against Staphylococcus aureus and other Gram positive bacteria, yeasts such as Candida albicans and fungi such as Trichophyton rubrum, responsible for athletes foot. Manuka oils which are rich in beta triketone compounds, appear to have the strongest antimicrobial activity.

The extent to which topical application of an extract of this plant can rival drug-based treatments at overcoming sores, was highlighted by a research project by two students at Whangaroa College in Northland recently. After hearing about a fellow student’s spider bite that wouldn’t heal until it was treated with a native plant preparation, the two students, Cheyenne Rush and Georgia Mills, decided to investigate the antibacterial properties of manuka essential oil and an extract of another native plant kawakawa (Macropiper excelsum).

Their experiment, which they entitled Te Rongoa Māori , involved collecting and growing colonies of bacteria, spreading these onto agar plates and applying a quarter of a teaspoon of each product to be tested. The relative rates of decline of the bacteria was recorded daily for 14 days, for the manuka oil and kawakawa extract preparations, in addition to the well known antiseptics Savlon® and Betadine®, which were applied to other agar plates as controls.

The results showed that manuka oil was the most powerful antibacterial, followed by Savlon® then Betadine®, with the Kawakawa leaf extract the least effective. Cheyenne and Georgia’s project thus showed that a simple, traditional plant preparation can be more effective than prominent antiseptic products in fighting wound colonising bacteria. It also won them a top prize at the recent Top Energy Far North Science and Technology Fair, which involved more than 150 participants from 10 schools in upper Northland(12).

Refs:

  1. Media Release from the Health Research Council, Soaring rate of skin infections linked to resistance.NZ Doctor, 20 September 2016.
  2. Williamson D et al, A bug in the ointment: topical antimicrobial usage and resistance in New Zealand. NZ Med J 2015; 128(1426):103-9.
  3. Upton A et al, Mupirocin and Staphylococcus aureus: a recent paradign of emerging antibiotic resistance. J Antimicrob Chemother. 2003; 51:613-617.
  4. Heffernan H et al, Demographics, antimicrobial susceptibility and molecular epidemiology of Staphyloccosu aureus in New Zealand, 2014. https://surv.esr.cri.nz/PDF_surveillance/Antimicrobial/Staph/2014Saureussurveyreport.pdf
  5. Antimicrobial Resistance: Global report on surveillance. World Health Organisation, who.int.ISBN 978 92 4 156474 8; (http://www.bbc.com/news/health-27204988).
  6. Williamson D et al, Missing in action: an antimicrobial resistance strategy for New Zealand. NZ Med J, 2015; 128(1427):65-67.
  7. Williamson DA, Hefferman H. The changing landscape of antimicrobial resistance in New Zealand. NZ Med J 2014; 127(1403):41-54.
  8. Walsh TR, The Lancet Infectious Diseases, 2016; 16(3): 23-33
  9. Carter DA, Front Microbiol 2016; 7:569
  10. Muller P et al, PLoS One 2013; 8(2):e57679
  11. Liu M et al, Front Microbiol 2015; 5:779.
  12. https://ssl-www.stuff.co.nz/auckland/local-news/northland/83909327/Manuka-proves-best-bacteria-fighter

Respiratory Health in Singapore and Herbal Options

I recently spent a couple of days in Singapore, where herbal product needs are currently somewhat different to those in my New Zealand home. September in Singapore generally marks the start of the 3-4 month so-called ‘Haze season’, a period in which the air can be tainted for days on end, with a haziness due to smoke drift from fires in nearby Indonesia. The annual haze season started early this year, in late August, and on 26 August Singapore’s 24 hour Pollutant Standards Index (PSI) entered the ‘unhealthy’ range of above 100, while its 3-hour PSI reached 215(1). As with the haze last year, when the PSI reading at times exceeded 300, most people didn’t venture out without a face mask.

Agricultural fires are an annual occurrence across Sumatra and in parts of Kalimantan on Borneo, as corporations as well as small-scale farmers use slash-and-burn methods to clear vegetation for palm oil, pulp and paper plantations. As well as trees and forests, there is much peat land in these parts of Indonesia, and peat fires can burn and smoulder underground for several months.

Tsmog-over-the-city-1197986-639x359he haze contains particulate matter, fine particulate matter, heavy metals and poly aromatic hydrocarbons, and at its peak can measure hundreds of kilometres across. As well as affecting Singapore’s air quality and visibility, the air pollution can spread to Malaysia, southern Thailand and the Philippines. This can have a major impact on the health of the people and plants of these countries, and of course those of Indonesia itself.

Fine particulate matter especially, can enter deep into the lungs, causing respiratory illnesses and lung damage. Particulate matter pollution and its constituents also damages plant morphological structure, flowering, water content, growth and reproduction, and can have genotoxic impacts(2). Epidemiological studies have shown an increase in morbidity and mortality rates from chronic obstructive pulmonary disease after exposure to elevated levels of air pollution, and associations between lung cancer and cardiovascular diseases, are well established.

This situation leads to increased demand for herbal lung health products in Singapore by the local population seeking to do more than wear a mask to protect their lungs against the damaging effects of the haze. Herbs that gently support and encourage the natural expectoration process of the millions of cilia cells lining our bronchial trees, whose role is to remove excess mucus and potentially harmful substances such as particulate matter or unwanted allergens, are therefore useful. These include mucilaginous (polysaccharide hydrocolloid rich) and expectorant herbs such as Marshmallow (Althaea officinalis), Mullein (Verbascum Thapsus) and the NZ native Hoheria (Hoheria populnea). Other traditional lung herbs such as Elecampane (Inula helenium), White horehound (Marrubium vulgare) and Hyssop (Hyssopus officinalis), are also useful. For Singaporeans having to live in the seasonal haze, or citizens of cities in China and many other countries where air pollution is a regular feature of life, in order to help protect against reduced levels of cellular oxygenation and an increased risk of bronchial congestion, asthma, lung cancer and heart disease, these herbs can be useful daily tonics.

In addition, certain herbs have chemo-preventive or protective effects against cellular damage and carcinogenicity, that may be helpful when exposure to air pollution is unavoidable. Apart from its anti-inflammatory, antimicrobial and antioxidant properties, evidence suggests a possible protective effects against lung cancer by roots of the warming volatile oil rich Elecampane(3).  The root of the fiery Horseradish (Armoracia rusticana)(4), and aerial parts of Nasturtium (Tropaelum majus),(5-6) also both contain phytochemicals with established chemo-preventive effects against cancers, that seem to be well absorbed into the bloodstream when taken orally. These and expectorant actions make them specifically indicated to help prevent lung damage in those exposed to regular dangerous levels of airborne pollution, such as the annual Haze in Singapore.

untitled-design-17While considering this situation, I couldn’t help notice the presence of palm oil still in chocolate sold throughout Singapore, unlike certain other countries where it has been removed due to public concerns around the environmental impacts of a huge increase in palm oil plantations. Similarly the importation of palm kernels for use as a supplementary feed to dairy cows in New Zealand, needs a mention. Reflecting on this as well as the widespread use of palm oil in cheap vegetable oils and in many other food and non food consumer items found globally, there is clearly a need to address the underlying cause of such environmental pollution and factors responsible for poor human health, in a more integrative way. This burning of indigenous forests in Indonesia is related also to poverty as well as poor regulation by authorities there, but corporate greed, consumer usage and lack of awareness or concern for environmental and economic impacts, is contributory.

Until the slash and burn method of land clearing in Indonesia is stopped, health effects on the millions of people living in the region, and ongoing widespread loss of bio-diverse rich forests and destruction of the habitat of endangered species such as orangutans, tigers, elephants and rhinos, will continue.

 

Refs:

  1. “The haze is back across South East Asia”. BBC. Retrieved 26 August 2016.
  2. Rai PK, Ecotoxicol Environ Saf 2016; 129:120-136.
  3. Li Y et al, Z Naturforsch C 2012; 67(7-8):375-380.
  4. Weil MJ et al, J Agric Food Chem 2005; 53(5):1440-1444.
  5. Platz S et al, Anal Bioanal Chem 2013; 405(23):7427-7436.
  6. Pintao AM, Planta Med 1995; 61(3):233-236.

Withania Improves Sexual Satisfaction in Women

Human sexual responses and functions are highly complex, with sexual desire and activities being influenced by multiple psychological, physical, environmental and relationship factors.

‘Sexual dysfunction’ is defined as difficulties that occur during the sexual response cycle that prevent the individual from experiencing satisfaction from sexual activity. While sexual dysfunction in men is relatively easy to characterise, female sexual responses tend to be more complicated, making measurement of appropriate contributory and outcome factors and the definition and treatment of female sexual dysfunction (FSD) more difficult (1).

Development of drugs such as sildenafil and others has made a big difference to the sex lives of men with erectile dysfunction over the past 15 years, but until now few drug treatment options exist for the most common sexual complaint in women, decreased sexual desire.

IMG_2611.CR2Ashwagandha (Withania somnifera) is a herb native to India and the Asian subcontinent, used as a tonic and treatment for a wide range of health conditions and perceived benefits. Known among herbalists as an ‘adaptogen’, its best known action is to help improve the body’s resilience to stress. Withania normalises blood levels of cortisol and other adrenal hormones during chronic stress, and exhibits a large number of actions suggesting an ability to insulate against adverse environmental stressors. This results in reduced anxiety, and a subtle but welcome retention of a sense of still being ‘in control’, during times of stress (2,3).

Withania is also used traditionally to treat men with erectile dysfunction and performance anxiety, and clinical studies have found improvement in sperm concentration and motility(4-6).  This reputation extends also to helping address diminished sexual desire in women, particularly where a depleted nervous system is contributory. A clinical study recently conducted in India, provides compelling data suggesting that it may well assist some women to achieve enhanced sexual satisfaction(8).

The study involved 50 women aged 21–50, all of who were in steady heterosexual relationships, and previously or presently engaged in sexual function for several years. Most were married women from affluent households who were not employed, but reported as having stressful lives due to social demands, child rearing, and husbands’ high expectations.

The women were divided into two groups, and both went through a counselling program consisting of two seminar presentations and an individualized consulting session on addressing FSD. One group also consumed capsules containing a 300mg extract of Withania (standardised to contain not less than 5% withanolides), and the other group placebo capsules, twice daily for 8 weeks.

The primary outcome measure was the Female Sexual Function Index (FSFI), a self-report 19-item questionnaire which was used at the beginning of the study, then at 4 and 8 weeks later(7). This included domain scores for desire, arousal, lubrication, orgasm, satisfaction, and pain. The FSFI Total Score was a weighted sum of these, and as expected was low at the study’s commencement.

Withania treatment lead to a significantly higher improvement than placebo in the FSFI, at both 4 and 8 weeks after starting the study. This increased from 13.63 to 23.86 in the treatment group, but only from 13.57 to 20.06 in the placebo group, a statistically significant difference. While mean scores for sexual desire or the number of total sexual encounters were similar in both Withania and placebo groups, mean scores for lubrication and orgasm were higher in Withania treated rather than placebo treated women. Women in the Withania treated group also reported a marked improvement in sexual satisfaction scores at 8 weeks, these increasing 1.4 points (from 2.35 to 3.79), versus only 0.36 points (from 2.34 to only 2.7) in the group of women who received counselling only (8).

Results from this study don’t suggest that Withania is an aphrodisiac, as Withania supplementation failed to statistically improve sexual desire or the number of total sexual encounters. However, the fact that Withania caused a marked increase in overall sexual satisfaction, in what was a relatively small clinical trial, offers considerable promise for women seeking a natural and safe herbal option for what is increasingly being recognised as a relatively common complaint in our modern world.

 

References:

  1. Chen CH et al, Taiwan J Obstet Gynecol 2013; 52(1):3-7.
  2. Cooley K et al, PLoS One 2009 Aug 31; 4(8):e6628.
  3. Rasmussen PL, Phytonews 39, Nov 2013; ISSN 1175-0251, published by Phytomed Medicinal Herbs Ltd, Auckland, New Zealand.
  4. Rasmussen PL, Phytonews 36, July 2011; ISSN 1175-0251, published by Phytomed Medicinal Herbs Ltd, Auckland, New Zealand.
  5. Pesch S et al, Theriogenology 2006; 66(2):307-313.
  6. Shukla KK et al, Fertil Steril 2009; 92(6):1934-1940.
  7. Rosen R et al, Journal of Sex & Marital Therapy, 2000; 26(2):191-208.
  8. Dongre S et al, Biomed Res Int 2015; 2015