Acute Oral Toxicity Evaluation of Hydroalcoholic Extracts of Saracaindica and Bauhinia variegata with the HPTLC -Fingerprinting and IR Characterization of Targeted Phytoconstituents

Introduction

Polycystic Ovary Syndrome (PCOS) is a highly common endocrine disorder in women of menopausal age. It is an intricate association of hormonal imbalances, metabolic disorders 5- 10% of women and, therefore, plays a major role in female infertility. The disorder usually occurs with abnormal or no menstual cycles, hyperandrogenism (elevated levels of male hormones), polycystic ovarian morphology as revealed in ultrasonography, and insulin resistance. All these physiological disturbs tend to translate into clinical changes of hirsutism, acne, obesity, alopecia and menstrual abnormalities. Along with reproductive complications, type 2 diabetes mellitus, cardiovascular disorde3rs, and endometrial hyperplasia or cancer are also the long-term health risks related to PCOS. PCOS is regarded as a hyperandrogenic disorder that is often followed by persistent oligo-ovulation or anovulation. Polycystic ovarian structure may be frequently determined with the help of ultrasonography or other imaging procedures, but not every woman with polycystic ovaries can have the clinical and biochemical conditions of PCOS. It has been reported that pregnancy prevalence of polycystic ovarian morphology has a range of between 20 and 33 percent in the general population. PCS women can exhibit a number of typical signs such as menstrual problems, obesity, hirsutism, acne, and changes in the biochemical parameters of leutinizing hormone (LH), testosterone, androstenedione, and insulin. Because of the complex nature and rising prevalence, there is need to create more awareness and research on the topic to enhance therapeutic interventions of managing PCOS.

The Precise etiology is unclear but, it is believed that a mixture of genetic predisposition, environmental factors and lifestyle factors are the cause of the etiology as hyperinsulinemia can stimulate excessive androgen secretion by the ovarian theca cells, which further worsens hormonal imbalance. The Rotterdam criteria are the most commonly used to diagnose PCOS by the presence of two or more of the following: oligo- or anovulation, clinical or biochemical hyperandrogenism, and ovarian polycystic or morbpholy on ultrasound.

The traditional treatment methods involve change in lifestyle, hormonal therapy (e.g., oral contraceptives), insulin-sensitizing drugs such as metformin and ovulation-inducing infertility medications. Nonetheless, more attention towards herbal medicines has been developing given their natural source and the possibility of fewer side effects that provides herbal medicines with a potential complementary option in the management of PCOS symptoms.

Materials and Methods

Plant Material and Extraction

Bark of Saracaindica and Bauhinia variegata were collected and processed. The plant materials underwent ethanol and aqueous(1:1 ratio) extraction using Soxhlet apparatus. Further HPTLC and IR has been done for the extract and their targeted phytoconstituents.

Preliminary Phytochemical screening

Standard qualitative texts for alkaloids , flavonoids  tannins and saponins has been done .

HPTLC fingerprinting

A CAMAG Llinomat-5applicator with a Hamilton microcrsyringe was used to apply bands in the chromatography analysis. The stationary phase was the use of pre-coated silica gel 60 F254 TLC plates.

FT-IR Analysis

Scanned under UV 254 nm and 366 nm. ATR spectra of extracts were captured in 4000- 400 cm-1 range of the functional groups that appeared as characteristic peaks on the instrument Shimadzu.

Experimental Animals

Animals were monitored during 14 days after the administration.Wistar rats of 190 ±10 g, 190 g were placed in a healthy condition in controlled laboratory conditions. The animals were put in a standardized environment and light and dark cycle was in a 12-hour cycle, temperature was maintained at 25 ±2 and relative humidity at 50-60. The ad libitum pellet diet and drinking water were administered during the period of study. All the experimental procedures were done in line with ethics, and they were approved by the Institutional Animal Ethics Committee (IAEC).

Acute Toxicity Study Protocol

The experiment was based on OECD guideline 423 ( Acute Oral Toxicity – Acute Toxic Class Method). The animals were assigned to four groups (n = 6 rats each group) and given one dose of extract Saracaindica and Bauhinia variegata at the rates of 5 mg/kg, 50 mg/kg, 300mg/kg, and 2000mg/kg orally.

Results

Preliminary studies of Extracts

 HE-SA: Hydroalcoholic extract of Saracaasoca, HE-BV: Hydroalcoholic extract of Bauhinia variegata,

+: Present, – :Absent

HPTLC fingerprinting of Saracaindica and Bauhinia variegata

HPTLC fingerprinting of Saracaindica

Application- Linomat-5 Applicator (CAMAG)

Sample Volume Applied- 20 µl

Solvent material- Chloroform: Acetone: Formic Acid =[75:16.5: 8.5]

Scan Wavelength- 254nm, 366nm

TLC Plate Development- Pre-saturated Twin Trough Chamber

ReferenceStandards:Catechin

Figure 1: Illustrate of HPTLC Plate at 254nm Click here to View Figure

Figure 2: Illustrate of HPTLC Plate at 365nm Click here to View Figure

Figure 3: Image of HPTLC Plate at White Light  Click here to View Figure

Table 1: Interpretationgraph  ofSaracaasoca and Catechin

Figure 4: Calibration curve of catechin Click here to View Figure

Figure 5: 3D-Plot at 254nm Click here to View Figure

HPTLC fingerprinting of Bauhinia variegata

Application- Linomat-5 Applicator (CAMAG)

Sample Volume Applied- 20 µl

Solvent System:-Toluene : Ethyl acetate : Formic acid (5:4:1)

Scan Wavelength- 254nm, 366nm

TLC Plate Development- Pre-saturated Twin Trough Chamber 

Figure 6: Image of HPTLC Plate of Apigenin at366nm and 254nm Click here to View Figure

Apigenin standard typically shows a sharp fluorescent yellow band under 366 nm.

Rfvalue of Apigenin: ~0.45–0.60 (depending on mobile phase system).Bauhinia variegata extract shows multiple bands due to mixed phytoconstituents.A band matching the Rf of apigenin standard confirms its presence in the extract.

Figure 7: Image of HPTLC Plate of extract and apigenin (fluorescent col.)at254nm and 366nm Click here to View Figure

FTIR Analysis

FTIR Analysis of  Saracaindica extract and catechin

Figure 8: IR spectra of Saracaasoca  Click here to View Figure

Table 2: IR interpretation of Saracaasoca and Catechin

Figure 9: IR spectra of Catechin Click here to View Figure

FT-IR Analysis of Bauhinia variegata extract and Apigenin

Figure 10: IR spectra of Bauhinia variegata  Click here to View Figure

Table 3: IR interpretation of Bauhinia variegata and Apigenin 

Figure 11: IR spectra of Apigenin Click here to View Figure

AcuteOral.Toxicity

Acute oral toxicity of Test sample Saracaindica 

Table 4: Body weight changes and Mortality

Table 5: Acute toxicity behavior changes of Saracaindica

Acute oral toxicity of Test sample Bauhinia Vareighata

Table 6: Body weight changes and Mortality

Table 7: Acute toxicity behavior changes of Bauhinia Vareighata

Discussion

The acute toxicity analysis of Saracaindica and bauhinia variegata plant extract in female Wistar rat species showed that there were no changes in its toxicity at the maximum dose of 2000mg/kg body weight. During the 14 days of observation, it was found that all the animals showed a slow and steady increase of their body weight, and there were no deaths in any group of doses. Additionally, close examination of behavioral and physiological parameters, including state of skin and fur, eye and mucous membranes looks, salivation, stool, sleep, somatomotor activity, and overall behavior revealed no deviations or any negative response. These results suggest that extraction of Saracaindica and bauhinia variegata does not produce any acute toxicity and has a high level of tolerance in animals when used in experimental studies.

The lack of mortality along with a considerable clinical effect even at the highest dose indicate that LD50 (lethal dose of 50 percent of the population) of extract of Saracaindica is higher than 2000 mg/kg, which makes this extract practically non-toxic in terms of OECD guidelines. This captive safety margin is especially necessary in the light of increased interest in herbal medicines as complementary or alternative therapeutic tools in the management of chronic endocrine disorders like Polycystic Ovary Syndrome (PCOS). PCOS may be a long-term condition, so it is important to determine the safety of herbal extracts in advance to avoid the possible adverse effects.

Saracaindica and bauhinia variegata extract has a high potential in the development as a therapeutic agent against PCOS based on its promising safety profile as evidenced in this study. Nevertheless, more research is suggested to be undertaken such as chronic toxicity research, reproductive toxicity research and in-depth pharmacodynamics and pharmacokinetics research. Such studies in the future will aid in assuring long term safety and efficacy which will open the way to clinical trials and a possible application of these therapeutic modalities in future in the management of PCOS.

Conclusion

This analysis showed that no acute toxic effects were observed with Saracaindica  and bauhinia variegata  extract at the highest dose of 2000mg/kg in female Wistar rats. The weight gain and lack of mortality and normal physiological and behavioral parameters indicate that the extract is practically non-toxic in acute exposure situations. This means that Saracaindica can be deemed safe to continue with further investigations on its chronic toxicity and pharmacological implications on the treatment of PCOS.

These results coincide with the other studies that suggested low toxic effect of related plant extract. This research contributes to the non-toxicity of bauhinia variegata and Saracaindica as therapeutic agents due to the current interest in the natural treatment of PCOS.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

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