In Vitro Investigation of Therapeutic and Anti-Coagulant Properties of Allium Sativum On Human Plasma

Cardio vascular illnesses are increasing worldwide and among these illnesses Thromboembolic disorders are very prevalent. The anticoagulant properties of many herbs have been reported. Garlic is a common herbal supplement. The current study was conducted to compare the in vitro anti-coagulant activity of different solvent-extracted fractions on human blood samples. For this purpose, rectified spirit and reverse osmosis (RO water), were used as solvents. Multiple extracting approaches were applied to prepare different extract fractions of Allium sativum , both in ethanolic and aqueous extracts by maceration, decoction, and soxhlet extraction methods. The concentration of each extract fraction was subjected to a primarily anti-coagulant screening method in human blood sample in-vitro by calculating their prothrombin time of coagulation. . The anti-coagulant activity of the extracts was determined by measuring the changes in prothrombin time with a null hypothesis value of p< 0.05. The results indicated that all garlic extract fractions had significant anti-coagulant potential. However, at 5 ppm concentration, soxhlet extraction extract had the maximum anti-coagulant potential. Moreover, Garlic's aqueous extract also showed a significant anti-coagulant effect on human plasma. This observation agrees with the obtained data that the soxhlet extracted sample of Garlic showed the highest activity of platelet aggregation inhibition. Both ethanolic and aqueous extracts of A. sativum showed significant anti-coagulation properties as compared to positive controlled EDTA and Double oxalate as a synthetic anti-coagulants in our study.


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With its medical value in mind, the current study focuses on the therapeutic and anti-coagulant effects of Allium Sativum on human plasma. As a result [23], the data was analyzed and categorized according to their relevance and a table was created to summarize all of the findings [24]. The findings are expected to add value in addressing concerns about the impact of garlic ingestion on blood pressure and cardiovascular morbidity [25].

Extract Preparation
Allium sativum L. ethanolic and aqueous extracts were prepared using rectified sprit and reverse osmosis (RO) water; their phytochemical analysis was conducted using the prepared stock standards. The collected sample was activated in its matured state by exposing it to sunlight under shadow for three days, for a total of eight hours per day. Following the color variation, samples were kept in the dark for 24 hours, then rinsed with purified water and packed in an air-sealed packet at -4 o C until the further procedure. UV spectroscopic and FTIR analysis on crude samples was conducted assess the qualitative analysis of Allium sativum. Phytochemical screening and proximate analysis was conducted as described by safowra [26], Trease and Millit [27] .
In this study, maceration, decoction, and soxhlet extractions were conducted to obtain Allium Sativum extracts at varied solute-solvent ratios (V/V). We used 1 mg of crude garlic extract in 100 mL rectified spirit to make a stock solution of 10 ppm that was concurrently diluted at 1 ppm concentration. In 10 distinct blood samples of 5 mL as whole blood, 1 ppm garlic extract was applied using phosphate potassium buffer for pH adjustment.

Collection of Blood Samples
Plasma samples were taken from healthy people according to a methodology that had been described earlier [28]. Healthy adults between the ages of 25 and 45 who did not have a family history of coagulation problems and were not on any medications met the inclusion criteria (e.g., thrombosis). The Mayo Hospital Lahore Ethics in Human Research Committee (#24124) and the Lahore Garrison University Ethics Review Board (LGUERB #02/20) both gave their approval to this study. Adult participants gave their informed consent to participate in the study.
Blood samples were collected in tubes containing 0.105 mol/L (i.e., 3.2%) in a ratio of nine volumes of whole blood to one volume of trisodium citrate an anti-coagulant. The prothrombin time test was performed according to the procedure described in the literature [29][30][31]. For appropriate coagulation investigations, the proper blood-to-anticoagulant ratio should be used.
According to NCCLS standards [32], the value of blood specimens hematocrits (HCT) should be about 55 percent for Prothrombin time screening i.e. 9 parts freshly collected whole blood and one part anti-coagulant should be used for each sample [33, 34].

Statistical Analysis
ANOVA with GraphPad Prism 7 software was used to examine differences in prothrombin time for an anti-coagulant impact of various garlic extracts, followed by post hoc Tukey's Multiple Comparison Test. Mean values were considered significantly different at P ≤ 0.05. All experimental data were reported as the mean ± standard deviation (SD) of analysis in triplicate using SPSS software.

Results
UV spectra of Garlic was examined in the wavelength range of 200-800 nm using Schimadzu double beam spectrophotometer. Absorption band at 220 nm is due to transition of valance electron in sulfonyl group [35,36]. We used an FTIR model number Nicolet 6700 to determine the major functional group in garlic extract at 400-4000 cm-1. The significant and larger peak at 3206 cm-1 in Figure 1 indicates the existence of a sulfonyl functional.

Phytochemical analysis
Allium Sativum are rich in nutrients such as protein, carbohydrate, minerals, dietary fiber and vitamins. These vitamins to play a significant role in preventing tissue damage because of free radicals, hence it is believed that Garlic is good source of antioxidants and anti-coagulant properties as shown in Table 1. Phytochemicals such as saponin, flavonoid, tannin, reducing sugar, steroid, and terpenoid were found in both aqueous and ethanolic extract of A. sativa.

Proximate Estimation
Proximate estimation is a semi-quantitative analysis used to evaluate the organic matter of samples and their accuracy in subsequent estimation analyses. We determine the organic and dry weight of the sample and estimate the nutrient and constituent ratio in the provided sample using proximate determination. To fulfil the criteria, this technique estimates moisture, protein, lipid, fat, and carbohydrates in the sample and compares them to standard statistics. This analysis employs a variety of methodologies and adaptations, but in present study, we used the preliminary method.
Three samples of data were collected and statistically analyzed to assess the outcomes. The data was provided in Mean Standard Error of Mean (n = 3) format. Table 2 shows the mean standard deviation (SD) analysis of all experimental data performed in triplicate using SPSS software (IBM, PASW 117 Statistics 254 19, USA).

Prothrombin Time and Anticoagulation Investigation
To investigate the anti-coagulant effects of Allium sativum extracts, a 5 ppm dose of garlic extract obtained using various extraction methods was used. The primary active dose with an anticoagulant therapeutical effect on humane blood plasma is garlic extract, extracted with maceration in an aqueous and soxhlet extractor. In a healthy person, the usual prothrombin time is 12-14 seconds. In a double-blind trial, we investigated the effect and efficiency of our Garlic crude extract using a positive controlled EDTA 5 mg combination and a negative control. To compare and verify the null hypothesis with garlic anti-coagulant action on humane blood plasma, we used the laboratory anti-coagulant chemical compound Ethylene diamine tetraacetic acid (EDTA) as a positive active control. Table 3 compares the anticoagulant effects of A. sativum L. (1ppm extract on humane whole blood to the other two controls groups). Table 4 shows the prothrombin time extrinsic route coagulation time in seconds, with two independent groups serving as positive and negative controls to verify anti-coagulant capabilities.
To test and verify the null hypothesis with garlic anti-coagulant activity on humane blood plasma, we included laboratory anti-coagulant chemical product Double Oxalate (combination of ammonium and potassium oxalate in the ratio of 3:2) as positive active dose.

Discussion
This study was carried on humane blood plasma that was extracted and isolated by centrifugation. However, any studies have reported the antithrombotic activity of raw garlic to be more effective than boiled product [48]. Another study has reported the in vitro effectiveness of aqueous extract of raw garlic to be more effective in comparison to the extract of boiled garlic against platelet aggregation [49].
The garlic extract that we prepared by the method of soxhlet extraction showed promising and better result than aqueous extract. The variance between all groups and validate our data significant we set significant p-value at P<0.05 and counter verified the groups significant and authentically rejected null hypothesis. Many studies have reported that depending on the type of solvent, extraction method and the plant used for extraction, the effectiveness of bioactive compounds in an extract can vary greatly [50].
In the second experiment, we examined the efficiency of garlic extract from both aqueous and ethanolic soxhlet extractions at 5 ppm against double oxalate as a positive control, and found that garlic extract at both 1 and 5 ppm concentrations surpassed the positive control group. However, when we compare the data from garlic extracts in aqueous solution with ethanolic extracts obtained using soxhlet extraction, we find that the ethanolic extract yields a better results. The standard deviation graph given in Figure 3 depicts the mean standard error among the random groups selected in the present study. Klebsiella) and gramme positive (S. aureus, S. pneumonia, streptococcus, and Bacillus anthrax) bacteria due to the presence of phytochemicals such as saponin and tannins. This study also discovered that A. sativum has a low fat content, and low fat diets are known to lower cholesterol levels. Low fat products have shown to reduce hypertension. Increased garlic consumption has shown to reduce hypertension [39,51].

Conclusion
The study was conducted to evaluate anticoagulant properties of aqueous and ethanolic extract of garlic on human blood. Among all the fractions of garlic extract tested in this study, ethanolic extract fraction had significant anticoagulant activity. The PT prolongation literally specified the inhibition of the extrinsic coagulation cascade. The phytochemical constituents of garlic can decrease fibrin formation. It was however not possible to determine which constituents in the extracts exactly were accountable for the activity. These herbal products are the symbol of safety in contrast to the synthetic drugs that are regarded as unsafe to human being and environment.
Although herbs had been priced for their medicinal, flavoring and aromatic qualities for centuries, the synthetic products of the modern age surpassed their importance, for a while. However, the blind dependence on synthetics is over and people are returning to the naturals with hope of safety and security. It is time to promote them globally.