Bhoj R Singh*
Principal Scientist and Head of Division of Epidemiology, India
*Corresponding author: Bhoj R Singh, Principal Scientist and Head of Division of Epidemiology, India
Submission: June 11, 2020;Published: July 17, 2020
ISSN: 2576-9162 Volume7 Issue5
Herbal antimycotics are being researched as antimicrobials to treat antibiotic-resistant fungal infections and as food preservatives (against food spoilage and mycotoxigenic fungi). Several antimycotic herbal compounds including cinnamaldehyde (in cinnamon, camphor, and cassia oils), carvacrol (in oregano, thyme, thymus and ajowan oils) and citral (in lemongrass, verbena, and citronella oils) inhibit yeasts and molds in microgram concentration. Their therapeutic use is still limited because of several biological, chemical, and pharmaceutical reasons. The dearth of long-term toxicity studies, pharmacodynamics and pharmacokinetics data, verifiable clinical trials data (at various stages of drug development), quality control, standard testing and preparation protocols, reference values and pharmacopoeia. Thus, proclaiming herbal antimycotics as future drugs either as an alternative or as a complementary therapeutic agent is still farfetched dream to come true.
Keywords: Herbs; Anti-mold; Anti-Candida; Drug-standards
Herbal antimicrobials are considered as the future drugs to fight against multi-drugresistant (MDR) microbes . However, reports of resistance in microbes to herbal antimicrobials  are common. Probably lack of systematic data leads to the misconception about unbeatable efficacy of herbal antimicrobials to counter MDR pathogens. Literature shows that many bacteria, yeasts and molds are resistant to antibiosis induced by herbal antimicrobials [3,4]. Although, scientific investigations claim efficacy of herbal antimycotics, they are not effective on all fungi (similar to antimycotic antibiotics) and thus, can only be used as an alternate or complementary to antibiotics as proposed for MDR bacteria .
Herbal antimycotics are classified based on their utility:
1) Inhibiting the environmental, plant pathogenic and food degrading fungi,
2) Inhibiting growth of potentially pathogenic fungi affecting human, animal and birds, and
3) Broad-spectrum ones inhibiting all different types of yeasts and molds. Another classification may be based on their activity as anti-yeasts, anti-molds, and broadspectrum affecting both yeasts and molds.
Anti-mold herbal antimicrobials
Molds are the source of many antibiotics and show resistance to a wide range of antibiotics as well as herbal antimicrobials. However, the molds that cause infections (invasive aspergillosis and mucormycosis) are also susceptible to selective antifungal drugs . Several herbal antimicrobials are found to be effective in in-vitro studies against molds causing infections. Essential oils of lemongrass (Cymbopogon citrates) [3,4], ajowan (Trachyspermum ammi) and African basil (Ocimum gratissimum)  inhibit 70-100% of pathogenic fungi; agar-wood (Aquilaria malaccensis) inhibit 65% of the pathogenic molds , carvacrol, an ingredient of essential oils of thyme (Thymus vulgaris), ajowan (Trachyspermum ammi) and oregano (Origanum vulgare) inhibit almost 80% to 100% of the pathogenic the molds , and cinnamaldehyde from cinnamon oil, cinnamodial and cinnamosmolide from Pleodendron costaricense kill majority of the molds , and many more, extensively reviewed by Vengurlekar and co-workers . However, essential oils of Aegle marmelos, Artemisia vulgaris, Caesulia axillaris, Cinnamomum tamala, Cinnamomum camphora, Citrus medica, Corymbia citriodora, Eupatorium cannabinum, Lippia alba, Mentha arvensis, Murraya koenigii, Nepeta hindostana, Citrus sinensis, Tagetes erecta, Vetiveria zizanioides, Vitex negundo, Zanthoxylum acanthopodium, Zingiber officinale , Commiphora mukul, Santalum album, Zanthoxyllum rhetsa and Pogostemon cablin  have weak anti-mold potential.
Besides essential oils, fixed oils of mustard with seeds of trigonella, ajwoin, mustard and garlic bulbs completely inhibited growth of Aspergillus niger, A. flavus, Absidia corymbifera, Penicilium nigricans and Candida albicans. On the other hand, coconut oil with ajowan seeds was less fungitoxic . Mycotoxin producing molds were inhibited with powdered herbs as cloves (Eugenia caryophyllus), cinnamon (Cinnamomum zeylanicum B.), allspice (Pimenta dioica), mustard (Sina.pis alba), garlic (Allium satiuum), and oregano (Oreganum vulgare) inhibited growth of mycotoxigenic molds at the 2% level in potato dextrose agar while thyme (Thymus vulgaris), turmeric (Curcuma longa), anise (Pimpinella onisum), paprika (Capsicum annum), red pepper (Red Cayenne, Capsicum annuum), black pepper (Piper nigrum), white pepper (Piper nigrum), leaves of sage (Salvia officinalis) and rosemary (Salvia rossmarinus) and onion (Allium cepa) had only little effect on the growth of mycotoxin producing fungi . Among the 56 Chinese herbs tested, decoctum (containing 10% of herbal extract by weight) from Carthamus tinctorius and Rheum palmatum effectively controlled the growth of A. flavus . In-vitro studies revealed the potential of herbal compounds as anti-mold. They have limited pharmaceutical value because most of the compounds may be toxic in therapeutic doses or their in vivo therapeutic concentration may be unachievable. Their utility as anti-mold is limited in food preservation due to unacceptable concentration required as preservative.
Anti-yeast herbal antimicrobials
Comparatively yeasts are more sensitive to antibiotics as well as herbal antimycotics than molds [4,13-15]. A variety of herbal compounds including essential oils of ajowan (Trachyspermum ammi), betel (Piper betle), guggul (Commiphora mukul), thyme (Thymus vulgaris), cinnamon (Cinnamomum verum and Cinnamomum zeylanicum), marjoram (Origanum majorana), holy basil (Ocimum sanctum), lemongrass (Cymbopogon citrates), sandalwood (Santalum album), Zanthoxyllum rhetsa, patchouli (Pogostemon cablin), thymus (Thymus villosus), peppermint (Mentha piperita), eucalyptus (Eucalyptus globulus), ginger grass (Cymbopogon martinii), coriander (Coriandrum sativum), agarwood (Aquilaria malaccensis), Eupatorium odoratum and Ageratum conizoides can restrict the growth or kill the yeasts [4,9,12,15,16]. The most potent anti-candida activity is reported in carvacrol (present in ajowan, oregano, thyme and thymus oils) and cinnamaldehyde (in cinnamon, camphor and cassia plant oils) that can inhibit almost all strains of yeasts in microgram concentration [4,15].
Besides essential oils, herbal extracts are also effective in controlling the growth of Candida albicans. Among effective herbal extracts, alcoholic extracts of Lawsonia inermis, Limonia acidissima, Tamarindus indica, Swertia chirata, Psidium guajana, Annona reticulate, Euphorbia hirta, Pogostemon parviflorus, Adenocalymma alliacum, Echinophora platybola, Cuminum cyminum, Withania somnifer, Curcuma longa, Cymbopogon citrates, and Zingiber officinale inhibited C. albicans at ≥5mg/mL concentration . Aloholic extracts of leaves of Ageratum conizoides and Eupatorium odoratum inhibited 20-40% Candida strains at ≥ 1mg/mL concentration . Although, the minimum inhibitory concentration (MIC) of alcoholic extracts for Candida albicans appears low, not low enough for therapeutic treatments . Testing 56 Chinese herbal drugs against Candida albicans; Scutellaria baicalensis and Rheum palmatum had the highest activity while against Geotrichum candidum extracts of Agastache rugosa and Pogostemon cablin were the best, but an extract of Mentha haplocalyx was only moderately effective but against all i.e., C. albicans, G. candidum and A. flavus .
Herbal compounds though promising as antimycotics, suffer from several deficiencies being included as mainstream antimycotic therapy. Herbal compounds like antibiotics vary in their efficacy and spectrum of activity as antimycotics. Some of the herbal compounds like cinnamaldehyde (in oils of cinnamon, camphor, and cassia), carvacrol (in oils of oregano, thyme, thymus and ajowan) and citral (in oils of lemongrass, verbena, and citronella) are considered broad spectrum antimycotics.
However, the biggest hurdle in use of herbal antimycotics in therapeutics are unavailability of verified and verifiable data on their toxicity in pharmaceutical doses, long term clinical safety and efficacy data and lack of pharmacodynamics and pharmacokinetics studies on herbal compounds. Besides, variability in the concentration of active ingredient and purity of active ingredient from batch to batch in herbs (as it depends on age, stage and season of harvesting herbs, and also on geographical location of their cultivation), variability even in in-vitro testing protocols and inconsistency in their reported efficacies, lack of pharmacopoeia standards for herbal antimicrobials, and problems in quality testing including lack of quality control standards and facilities are still bigger problems. Therefore, only through mending deficiencies mentioned herein herbal antimicrobials can be seen as future drugs either as an alternative or as a complementary therapeutic agent.
If not otherwise stated, all chemicals and reagents were purchased from Sigma l (St. Louis, MO), with the exception of fetal bovine serum (FBS), which was purchased from Gibco BRL (Paisley, Scotland, UK).Animal and OPU
The study was conducted in a farm located in the veterinary staff of the Division of Animal Resources of Guangxi Buffalo Research Institute (GBRI) Chinese Academy of Agricultural Sciences (CAAS).Ovum pick-up was carried out healthy, multiparous and lactating buffalo cows (Murrah, Nili-Ravi and cross-breed buffalo) with 92 heads. The donors were under controlled nutrition, barnhoused, and restrained in a chute at the moment of the oocyte retrieval session, then prepared for follicular as described by the reporters [5,7,13] for buffaloes. HS2000 Ultrasonic imaging produced by Honda of Japan, with 7.5/5.0MHz vaginal sector scan probe (Aloka, SSD-500, Tokyo, Japan) equipped with 55cm long needle for oocyte collection, vacuum pumps (Cook IVF Co., Australia), follicular fluid collection tube, temperature devices. The number and size of follicles in each ovary was determined before puncture. Follicles of ≥3mm in diameter were punctured and the follicle contents were drawn into the 50-ml Falcon tube using a regulated vacuum pump at 50mmHg vacuum pressure. During follicle aspiration the aspiration line was continuously rinsed with aspiration medium. OPU was conducted once every three days intervals and performed five times continuously.COCs processing
Aspiration medium consisted of DPBS with addition of 3% fetal bovine serum (FBS) and 100μg/ml, penicillin and 60mg/ml streptomycin and 10IU/ml heparin. The rinsed TCM199 medium contained 5% NBS. According to the morphological characteristics of cumulus cell complex (COCs), the oocytes were classified according to the method described by Zhang Xiufang et al. .
Cumulus-oocytes complexes (COCs) were classified in 4 categories.
The average number of oocytes obtained by OPU and the rate of high-quality oocytes were evaluated. SPSS 17.0 software was used for statistical analysis. With different batches as fixed factors, the number of follicles was regarded as dependent variable and the number of follicles was regarded as dependent variable. Single factor analysis of variance (One-Way ANOVA) was used to test significance. The difference was significant by Duncan’s method, using P<0. 05 as the criterion of significance.
The results showed that the average percentage of high-quality oocytes (A and B grade) was 66.05%. When conducting continuous LOPU, the rate of high quality oocytes (grade A)from the first OPU to the fifth one were 58.95%, 64.05% and 70.28%, 69.07% and 67.86%, respectively, while the recoveries rate were 43.07%, 35.43% 4.60%, 37.42% and 37.46%respectively,both showed no significant difference between each batch (P>0.05).However, the rate of grade C oocytes (naked eggs and reticular cells) during 5 times were 39.99%, 24.65%, 15.95%, 23.81% and 21.17%, respectively, showed a significant difference (P< 0.0001), and with a decreasing trend as the frequency of LOPU increases.
A literature  reported that although there were differences in the average number of punctured follicles and the number of oocytes recovered from every donor successive three times of OPU, there was no difference in the number of oocytes available to each head. In this study, the quality oocytes of grade A and B were the lowest in the first OPU, the second time was increased, the fourth time was relatively stable, but there was no significant difference among the batches of OPU. However, the changes of C grade oocytes were significantly different among batches (P<0.0001). With the increase of the number of OPU, the percentage of C grade oocytes (denuded oocytes and reticular cells) decreased continuously. However, there was no significant difference in the percentage of high quality oocytes and the recovery rate among the batches (P> 0. 05). The average percentage of high quality oocytes of grade A and B grade were 66. 05% and was similar to the result which 70.92 % . Another the recovery rate of oocytes was 71.60% in Murrah buffalo which were treated with eCG . Furthermore, average recovery of no hormonal treatment buffalo oocytes had the similar result (64.18%).
As for the recovery rate of OPU in buffalo,  reported that oocyte availability rate in the first OPU was only 56.20%, and the first oocyte availability rate in this study was 58.95%, the results were very similar. But the availability rate of the 2nd and third OPU (64.05±3.63%, 70.28±3.82%) was lower than that of Liang Xianwei (80.98%, 80.60%), which might be different from the variety, quantity and season of the donor, nutrition and other factors related. But the incidence of good quality oocytes (Grade A +B COCs) was also not affected by season in Mediterranean buffaloes . The reason for the low availability of oocytes in the first oocyte collection may be that there are more atresia follicles at the first time than at other times. It was agree with the point of view that higher level of follicular atresia was reported  and, consequently, a lower number of total recoverable and viable oocytes. With the increase of the number of oocytes collected the number of the atretic follicles decreases and the quality of the oocytes increases. As a result, the proportion of available COCs recovered increased . The results also showed that the quality oocytes remained relatively stable at the fifth time. However, there was no significant difference in the recovery of oocytes. However, the changes of C-grade oocytes in different batches were significantly different (P< 0.0001). With the increase of the number of OPU, the percentage of C-grade oocytes (denuded oocytes and reticular cells) decreased continuously. The average rate of denuded oocytes recovered in this study was 25.15%, which was higher than that reported by Liang Xianwei et al.  (16.33%), but lower than that reported by Yadav et al  (39.9%) Figure 1 and Table 1.
Figure 1: Effect of OPU replicate on oocyte quality.
Table 1: Effect of OPU replicate on oocyte quality (Mean±S.E.M).
OPU replicate effected on oocyte quality. With the increase of the number of OPU, the number of high quality oocytes (GradeA+B) increases. There was significant difference in Grade C.
This research project is supported by Guangxi key R & D Program (Gui Ke AB 16380040) and Guangxi Science and Technology Development Project (Gui Ke AD19259009) and State Key Laboratory for Conservation and Utilization of Subtropical Agro-bio resources ,Guangxi University (SKLCUSA-b201814).
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