Effect of Aqueous Extract and Fractions of Fagonia arabica on In Vitro Anticoagulant Activity

Introduction

Anticoagulants have been widely used in both clinical and in vitro medical treatments. In clinical practice, they are the drugs of choice for the prevention and treatment of thromboembolic disorders and prophylaxis of thrombotic events both pre- and postsurgery. It is estimated that of 1000 patients, only 2 to 4 receive anticoagulant therapy. ^1,2 Substances with anticoagulant activities are among the first ones to chosen as functional components that are being used to open up new areas of application for anticoagulants.

Anticoagulant monotherapy has been the basis of treatment for patients with atrial fibrillation and left ventricular thrombus as well as for prevention or treatment of deep venous thrombosis and pulmonary embolism. Considering the adverse effects of the existing anticoagulant therapy, alternative drugs from natural origin can help get the new molecular lead as a hope toward the better efficacious and safe anticoagulant.

Modern pharmaceutical industries are researching to identify new active components, and they can be either natural or synthetic used for curing several diseases. Worldwide, about 30% of the pharmaceuticals are prepared from plants. ³ Epidemiologic studies have provided evidence that foods with experimentally proved antithrombotic effect could prevent coronary events and stroke. ⁴ Considerable efforts have been directed toward the discovery and development of natural products from various plant and animal sources that have antiplatelet, ^5,6 anticoagulant, ^7,8 and antithrombotic ⁹ activity.

Fagonia arabica (FA) belongs to the family Zygoplyllaceae. ¹⁰ The FA is a small spiny undershrub with stiff, more or less prostrate, branches and found in northwest India and Deccan. The leaves of FA are arranged opposite to each other, with 1 to 3 foliolates, entire, linear-elliptic leaflets, and small, pink flowers. ¹¹ Since ancient times, FA has been reported to be used as a folk medicine for the remedy of various ailments. Uses of FA as blood purifier and deobstruent were also mentioned in Ayurvedic description. ¹² In recent times, FA was reported to possess antioxidant properties, ¹³ thrombolytic, and ¹⁴ mollucidal activities ¹⁵ and has also been used for cancer treatment and in liver empowerment. ¹⁶ We undertook the present study to evaluate the potential antithrombotic effects of the extracts of FA and its derived fractions.

Materials and Methods

Results

Effect on Plasma RT by Multisolvent Extract of FA

The in vitro anticoagulant properties of the multisolvent extract (MSE) were assessed by RT assays using healthy human plasma (Figure 1). The normal value of RT for plasma from a healthy human was 4.2 minutes. The RT was prolonged to 6 minutes at a concentration of 1 mg/mL. Increasing the concentration of the extract led to an even higher prolongation of RT. This shows that a concentration-dependent anticoagulant activity was obtained. At 20 mg/mL concentration, the time taken for clot formation increased to approximately 32 minutes. The result was statistically significant as the P value was <.0001 when time taken for coagulation by FA was compared with that of saline (control). With heparin as positive control (0.9 µg/mL), 38.5 minutes of prolonged coagulation time was observed.

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Figure 1.

Anticoagulant effect of multisolvent extract (MSE) of Fagonia arabica (FA), positive control (heparin) and negative control (saline) by plasma recalcification assay (n = 20). Values are expressed as mean ± standard deviation. Significance was determined by 1-way analysis of variance, followed by Duncan multiple range t tests. The results were statistically significant as P < .001.

Plasma Recalcification Assay of FA Extracts Prepared With Different Solvents

Among all the extracts assayed for anticoagulation, namely, water (aqueous), ethanol, methanol, IPA, butanol, ethyl acetate, acetone, chloroform, petroleum ether, and hexane, aqueous extract was found to be most effective as it delayed the RT up to 27 minutes, which is equivalent to the MSE extract of FA, that is, 27.34 minutes (n = 10; P < .001). Anticoagulant effects of all the extracts are tabulated in Table 1. Negligible activity was observed with other extracts (Table 1).

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Table 1.

Anticoagulant Effect of the 10 Extracts Prepared by Successive Extraction, Multisolvent Extract (MSE) of FA, Heparin, and Saline by Plasma Recalcification Assay (n = 10).

S.No.	Drug/Herb Extract	Time (minutes)
1	Heparin	34.4
2	Saline	3.96
3	FA (MSE)	27.34
4	FA aqueous	27
5	Methanol (MeOH)	4.7
6	MeOH control	0
7	Ethanol (EtOH)	3.7
8	EtOH control	5.6
9	Iso-propyl alcohol (IPA)	7.1
10	IPA control	0
11	Butanol (BuOH)	6.2
12	BuOH control	5.6
13	Ethyl acetate	9.4
14	Ethyl acetate control	9.5
15	Acetone	4.5
16	Acetone control	7
17	Petroleum ether	5
18	Petroleum ether control	0
19	Hexane	6.38
20	Hexane control	0

Abbreviation: FA, Fagonia arabica.

Preparation of FA Fractions

Thin-Layer Chromatography

Both filtrate portion and solid mass of FA were subjected to TLC as described earlier. According to the TLC analysis of the ethanol soluble filtrate, 5 spots were detected (n = 10), and the retardation factor values of each spot are listed in Table 2. However, the TLC of the solid portion showed only a single spot (Table 2).

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Table 2.

Results of the Filtrate and Solid Mass of Aqueous Extract of FA Obtained by Thin-Layer Chromatography (TLC).

Serial No.	Spot	Rf Value
1	Rf Spot 1	0.97
2	Rf Spot 2	0.84
3	Rf Spot 3	0.63
4	Rf Spot 4	0.13
5	Rf Spot 5	0.00
Solid mass	Rf A	0.47

Abbreviation: Rf, retardation factor.

Column Chromatography

After TLC, each spot was separated and collected with the same eluent system as that of TLC. With chloroform–ethanol (1:1), 4 spots were collected and stored at 4°C for further analysis. For the collection of the fifth fraction, the eluent system was changed to ethanol–distilled water (3:7). The fractions thus collected were named as FA1, FA2, FA3, FA4, and FA5.

Effect of FA Fractions on In Vitro Anticoagulation Activity

All the 5 fractions collected by TLC-guided column chromatography of FA (aqueous) were subjected to plasma recalcification assay (Figure 2). With FA1, FA2, FA3, and FA4, 6.7, 9.92, 7.11, and 7.58 minutes of anticoagulation was noted, respectively. Maximum anticoagulation was found in FA5. The FA5 of 100 µL was equally effective as heparin (20 µg/mL), with 25.16 minutes of RT (P < .001).

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Figure 2.

Anticoagulant effect of fractions of aqueous extract of Fagonia arabica (FA; FA1, FA2, FA3, FA4, and FA5) with heparin as positive control and saline as negative control (n = 10). Values are expressed as mean ± standard deviation. Significance was determined by 1-way analysis of variance, followed by Duncan multiple range t tests. The FA5 was found to be most active for anticoagulation and the P value was <.001.

Phytochemical Screening of FA5

The FA5 was estimated for the presence of the following phytochemicals: flavonoids, carbohydrate, reducing sugar (monosaccharide and polysaccharide), alkaloids, protein, tannin, triterpenoid, and saponin. The most active fraction (FA5) was positive for flavonoids, carbohydrate, reducing sugar, monosaccharide, tannin, terpenoid, and saponin (Table 3). Total yield of FA5 was 20.82% extracted from 2 kg of dried FA.

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Table 3.

List of Phytochemicals Identified in the Fifth Fraction of Aqueous Extract (FA5).

Phytoconstituent	Fifth Fraction of Aqueous Extract
Flavonoid	+
Carbohydrate	+
Reducing sugar	+
Polysaccharide	−
Monosaccharide	+
Tannin	+
Triterpenoids	+
Saponin	+
Alkaloid	−

Discussion

Anticoagulants are agents that prevent the clotting of blood. A thrombus is formed only in in vivo (unlike blood clots that can form in vitro). Pieces of the thrombus may break off and form an embolism that may lodge in vessels in the lungs or brain or any other body part, causing damage to the tissues supplied. Thrombolytic drugs are able to dissolve thrombi, and the antiplatelet drugs diminish the adhesion of platelets and their contribution to thrombus formation. In some situations, for example, myocardial infarction, the 3 classes of drug—anticoagulants, antiplatelets, and thrombolytics—may be used in combination. ²⁰

Antithrombotic, thrombolytic, and antiplatelet drugs are used to cure the patients with atherothrombosis. Through our experiments, it was found that FA possesses thrombolytic property that is proved both by an in-house developed Tube method of Central Indian Institute of Medical Sciences ²¹ and by Microtitre Plate Clot Lysis assay. ²² Moreover, its antioxidant property was recently reported, and its neuroprotective role was defined. ¹³ Anticoagulant property of FA was not studied so far; therefore, it was desirable to study the anticoagulant property of FA. Hence, anticoagulation property of FA has been studied by plasma recalcification assay along with partial characterization of FA.

The MSE of FA purchased from Innocon Foods was tested for anticoagulation by plasma recalcification assay and compared with positive (heparin) and negative controls (saline). With saline, it took 4.2 minutes to form a firm clot; however, with heparin the time taken for firm clot formation increased to 38.5 minutes. Interestingly, with increasing concentration of MSE, the time for clot formation also increased in increasing order. The highest anticoagulation activity was found with 20 mg/mL concentration of MSE that is 31.5 minutes (Figure 1). With repeated experiments, the same results were observed; thus, it was clear that MSE of FA possesses anticoagulation property in vitro. In the next phase of this study, in order to characterize the potential effect of FA, 10 raw dried FA extracts were prepared successively with increasing polarity of solvents. Aqueous extract was the most potent extract (Table 1) possessing the anticoagulation property, as its effect was almost similar to that of FA extract purchased commercially as well as with the positive control heparin. With other extracts, negligible activity was noted; thus, aqueous extract was chosen for further characterization.

Aqueous extract was reduced by rotary evaporator. It was then subjected to a series of steps including TLC and separation of collected spots by column chromatography. All the 5 spots/fractions (FA1-FA5) thus collected were subjected to in vitro anticoagulation assay to determine the most active anticoagulation fraction. Interestingly, FA5 was found to be the most potent anticoagulant fraction (27 minutes) that was very near to the effect noted with commercial FA extract (31 minutes). Thus, we can say that FA5 contains the most active component responsible for the anticoagulation effect.

To analyze the contents of FA5, it was subjected to phytochemical screening. The FA5 was found positive for saponin, flavonoids, tannin, triterpenoids, carbohydrates, monosaccharide, and reducing sugar.

Through this study, it was made clear that FA possesses anticoagulant property; and after partial characterization, it was further demonstrated that FA5 possesses the active anticoagulant property and contains several phytochemicals. Plasma recalcification assay is the most convenient method for general anticoagulation testing of newer drugs/herbs. ^23,24 This test is convenient to perform and requires no analyzer or special instrument to evaluate anticoagulation. To the test plasma, CaCl₂ is added and observed for coagulation by measuring the time taken for clot formation. The cost of the test is minimal and is good to study anticoagulation of newer test drugs. The anticoagulation effect can be studied in general, but there are other methods that could be used for further determination of anticoagulant mechanism, for example PT, APTT, PTT, and so on. ^25,26 These assays test the intrinsic and extrinsic coagulation cascade of the newer drugs. These tests are used worldwide to study anticoagulation; however, recently 3 new tests are reported to study the intrinsic and extrinsic coagulation pathways with respect to newer drugs/herbs. These are The intrinsic coagulation activity assay (INCA), The extrinsic coagulation activity assay (EXCA), and The recalcified coagulation activity assay (RECA). ^{27 –29} All the 3 methods are recent global assay used to study coagulation, are more advanced techniques that require less reagents (than PT, APTT and PTT assay), and require no special analyzer to identify and calculate the coagulation time with respect to newer anticoagulant drug study in individual patients. For financially unaided laboratories, installing new analyzers or instruments and bearing the cost of reagents are difficult; hence, plasma recalcification assay is the most convenient and affordable assay to study anticoagulation initially for newer drugs. As with FA, we have found significant anticoagulation effects, and FA can be further studied using INCA, EXCA, and RECA in future for better understanding of the mechanism.

The FA as a whole or its fraction could be investigated for anticoagulant activity using in vivo experimental protocol. Further studies could also reveal whether phytoconstituents of FA5 are responsible for anticoagulation. As FA is an herb and is used for medication since ancient times, the related side effects could be negligible. Hence, FA could be used as a new effective herbal anticoagulant for the treatment of patients with thrombosis and associated diseases. Through earlier studies conducted by other researchers, it was reported that FA possesses the following components: flavonoids (6 types) isorhamnetin 3-glucoside and 3-rutinoside, herbacetin 8-rutinoside, herbacetin 8-methyl ether-3-rutinoside, 3,7-diglucoside, and 3-rutinoside-7-glucoside, triterpenoidal glycosides, sulfated triterpenes, sulfated triterpene glycosides, saturated and sulfated pentacyclic triterpene of the taraxastane series with a C-20, 28 lactone unit, monodesmosidic saponins, bidesmosidic saponins, ceryl alcohol,-sitosterol n-triacontanal chinovic acid, fagogenin, saponin, genin A genin B, stigmasterol, campesterol, and harmine. Three triterpenoid of oleanane group, triterpenoid saponin mixture containing saponin A, B, and C, 5 substituted 8-methoxyflavones, and 2 erythroxan-type diterpene, fagonone (16-O-acetylflagonone), and minerals, namely, Zn, Na, Mn, Fe, P, K, Ca, Cu, vitamin C, protein, amino acids (alanine, arginine, glycine, isoleucine, leucine, lysine, phenylalanine, proline, tyrosine, valine) were also found. ^{15,30 –33} Through characterization of the phytochemicals identified in FA5 with reference to the reported phytochemicals, the most active component could be identified and analyzed for anticoagulation.

Anticoagulants play a pivotal role as agents for the prevention and treatment of thromboembolic disorder. ^34,35 For more than 5 decades, anticoagulant such as heparins, vitamin K-antagonists, and their derivatives have been the major players in the clinical practice. Although their efficacy remains undisputed, the fatal life-threatening side effects of these drugs have also been well documented. ^36–37 The cost of developing a novel anticoagulant is also given due consideration, whereby a cheaper yet effective alternative would be of immense value. ^38,39 In addition, there is a pressing need for an orally available anticoagulant agent to replace warfarin. ⁴⁰ At present, the focus is on potent anticoagulant sources hailing from natural origin. There is a compelling need for the consumption of dietary anticoagulants or phytochemicals with anticoagulant properties, which can ultimately reduce or eliminate the risks of thromboembolic diseases. ⁴¹ Hence, anticoagulants of natural origin provide a good remedy/therapy for the aforementioned disorders. ⁴² Moreover, anticoagulants have also been identified from other natural sources such as cellulose sulfate, ⁴³ sulfated flavonoids, ⁴⁴ halothurian glycosaminoglycan, ⁴⁵ a glycosaminoglycan extracted from the body wall of the sea cucumber, ⁸ protein from Taraxacum platycarpum, ⁴⁶ and many more.

The hot water extraction of the whole plant of FA gave the highest yield and exhibited the most potent anticoagulant properties for all parameters tested in vitro. These results highlight the biochemical nature of the active compounds in FA that might contribute to anticoagulation. Since it is evident that hot water has the ability to extract the phytochemicals responsible for the activity, presumably, the active compounds extracted at this temperature are thermally labile. Many traditional medicinal plants and herbs have been reported to have various levels of anticoagulant activity. In general, plant extracts of FA have yielded promising results, hence it seems reasonable to conclude that there are probably important anticoagulant compounds present in FA, but we need to isolate and identify these active constituents. Further characterization will reveal the identity of these compounds. Hence, it could be stated that the phytochemicals thus identified in this study could be responsible for anticoagulation action of FA otherwise FA as a whole extract could also be incorporated in specific preparation for anticoagulation.

The physicians give both thrombolytic and antithrombotic drugs for patients with thrombosis. This type of combination ensures that thrombolytic drugs would dissolve the clots already formed in the veins and arteries and anticoagulant drug will prevent further clot formation. In lieu of the present study and with our earlier findings, FA possesses both thrombolytic and anticoagulant activities. There are other natural products and their components that possess both anticoagulant and thrombolytic properties such as onion, ⁴⁷ Paeonia anomala extract, ⁴⁸ tuftsin, ⁴⁹ Paeonia lutea root bark, ⁵⁰ danshen (Salvia miltiorrhiza), ⁵¹ and meadowsweet (Filipendula ulmaria (L) Maxim.). ^{52 –54}

The FA could be a better agent for curing patients with thrombosis, as it would act as both a clot dissolver and an anticoagulant agent. Moreover, being a natural product, the related side effects would be less and therefore safer.

Conclusion

Fagonia arabica extract (aqueous and commercial) and fractions of aqueous extract (obtained by column chroma-tography based on TLC-guided results) were evaluated for anticoagulation activity based on an RT assay in an in vitro system. Prolongation of RT produced by the FA and its fractions increased with an increase in the concentration of FA. Further investigations are needed to isolate the bioactive compounds responsible for the anticoagulant activity as well as for the determination of the coagulation factors affected. This natural source is a cheaper and better alternative anticoagulant because of the abundant availability of raw material and simple extraction methods.