Herbal Antimycotics and Their Prospects in Therapeutics

Prospects

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).

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.

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. [14].

Cumulus-oocytes complexes (COCs) were classified in 4 categories.

1. Grade A (oocytes with more than two layers of cumulus cells and homogenous cytoplasm);
2. Grade B (oocytes with at least one layer of cumulus cells and homogenous cytoplasm);
3. Grade C (oocytes partially denuded, but still showing homogenous cytoplasm);
4. Grade D (degenerated oocytes with irregular shrunken cytoplasm). Grade A and B COCs were cultured in vitro maturated medium (TCM199+ 10% FBS+ 10µg/mL FSH + 12 U/mL LH + 1 µg/mL E2+ 50 µM cysteine) under a humidified atmosphere of 5% CO₂ at 39°C for 22 to 24 h(Pang et al, 2009)

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 [15] 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 % [15]. Another the recovery rate of oocytes was 71.60% in Murrah buffalo which were treated with eCG [16]. Furthermore, average recovery of no hormonal treatment buffalo oocytes had the similar result (64.18%).

As for the recovery rate of OPU in buffalo, [15] 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 [17]. 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 [18] 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 [19]. 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. [15] (16.33%), but lower than that reported by Yadav et al [16] (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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