Phytochemical screening, antibacterial and antioxidant activity studies on the crude root extract of Clematis hirsuta

Abstract

Clematis hirsuta in the family of Ranunculaceae is geographically dispersed in highland areas including Ethiopia. The leaves and roots of Clematis hirsuta is traditionally used to treat various diseases in different regions of Ethiopia. This study is aimed to evaluate the phytochemical constituents, antibacterial and antioxidant activity studies on the crude root extracts of Clematis hirsuta. The air-dried plant sample was powdered, extracted successively with n-hexane, chloroform and methanol. The solid matter was separated by filtration and then filtrates were concentrated using a rotary evaporator. The dried extract was subjected to a preliminary phytochemical screening test using standard procedures and the result showed the presence of alkaloid, saponins, tannins, flavonoids, phenols, glycosides and carbohydrates. Antibacterial activity of the crude extract was determined by using the disc diffusion method. The methanol extract of the roots of Clematis hirsuta showed a significant inhibition zone on all of the selected bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella thyphi) while chloroform extract had inhibition on Pseudomonasaeruginosa and Staphylococcus aureus. The methanol crude extract was screened for antioxidant activity by the DPPH assay method. At 2 mg/mL methanol crude extract gave %DPPH inhibition of 98.2% as compared to reference ascorbic acid (95.8%) at the same concentration. This study showed that the roots of Clematis hirsuta constitute phytochemicals that possess antibacterial and antioxidant activities and can serve as a potential source in search of plant-based antibiotics and natural antioxidants.

Keywords:

• Clematis hirsuta
• crude extract
• phytochemical screening
• DPPH

PUBLIC INTEREST STATEMENT

According to WHO, nearly 80% of the world's population relies on the use of traditional medicines to meet their primary health-care needs. Plants are also the basis for the development of modern drugs. They produce secondary metabolities like flavonoids, terpenoids, phenolic compounds, and saponins which constitute antimicrobial and antioxidant properties. Clematis hirsuta is one of the medicinal plants used in Ethiopia to treat different kinds of diseases like blackleg, respiratory tract problem and cataract, edema, leishmaniasis, herpes, hemorrhoids and tumor on the neck, among others. Powder made from barks, leaves and stems of the plant is applied directly to affected tumor sites. It is from this background that the researcher tried to screen the phytochemical constituents, and test the antioxidant and antibacterial activities of the root extract of Clematis hirsuta. The findings showed moderate antioxidant and antibacterial activities of the plant extract. In addition, phytochemicals like alkaloids, saponins, flavonoids, phenols, tannins, glycosides and carbohydrates were observed.

Competing interest

The authors declare no competing interests.

1. Introduction

Plants are the basis for the development of modern drugs for many years in daily life to treat diseases all over the world (Ates & Turgay, 2003). Traditional societies in Africa and elsewhere have always used herbs to promote healing (Bussmann, 2006). Many of the plant materials used in traditional medicine are readily available in rural areas at relatively cheaper than modern medicine (Mann et al., 2008). Plants generally produce many secondary metabolites which constitute an important source of microbicides, pesticides and many pharmaceutical drugs. Currently, there is growing interest, both in the industry and in the scientific research, for medicinal plants because of their antimicrobial and antioxidant properties. These properties are due to many active phytochemicals including flavanoids, terpenoids, carotenoids, coumarone, curcumines, etc. Plants have provided a good source of anti-infective agents; emetine, quinine, berberine, tannins, terpenoids, alkaloids and flavonoids remain highly effective instruments in the fight against microbial infections (Cowan, 1999). Nearly 80% of the world's population relies on the use of traditional medicines to meet their primary health-care needs. Out of the total 422,000 flowering plants reported from the world, more than 50,000 are used for medicinal purposes (Hamilton, 2004).

Clematis hirsuta (C. hirsuta) is a species of woody climbing plant (Figure 1) among more than 250 species in the family of Ranunculacae. It is found widely spread in tropical Africa and regions of intermediate altitudes, including Ethiopia. In Asia, Clematis hirsuta is used in folk medicine as anti-inflammatory, analgesics and anti-rheumatics (Al-Taweel et al., 2007). Clematis hirsuta with the local name “Hidda Feetii” is one of the medicinal plants used in Ethiopia to treat different kinds of diseases. As reported by Yineger et al. (2007), roots of C. hirsuta and Sida schimperiana (Malvaceae) are crushed, powdered and mixed with water for oral and nasal administration to treat blackleg and for the treatment of various animal ailments in Bale Districts. Clematis Hirsuta is also used to heal respiratory tract problem and cataract in the Meinit ethnic group, the southern part of Ethiopia (Giday et al., 2009).

Habtamu and Mekonnen (2017) reported that the 80% methanol and chloroform extract of the leaves of Clematis hirsuta showed moderate inhibition zone on some pathogenic bacteria. The chloroform crude extract of the leaf Clematis hirsuta showed a better inhibition zone (12.33 ± 0.50) on P. aeruginosa at 200 mg/mL concentration. The lowest MIC and minimum bactericidal concentration (MBC) of the chloroform extract of Clematis hirsuta reported by this group were 3.125 and 6.25 mg/mL on P. aeruginosa, respectively.

Due to these traditional medicinal uses and other applications of Clematis hirsuta, this study has given attention to identify secondary metabolities present in the root of the plant and evaluate its antibacterial and antioxidant properties.

Figure 1. A) Aerial part; B) Root of Clematis hirsuta (Picture taken by Fekede Kenea in June, 2018)

2. Materials and methods

2.1. Plant sample collection

Fresh roots of Clematis hirsuta were collected from the open fields of Nefuro Dini area of Sirba Wadessa kebele, in Gida Ayana district of East Wollega Zone in July 2018. Taxonomic identification was done and a voucher specimen (ah004) was deposited at plant pathology (biology department) of Wollega University. The fresh root of the plant was washed with tap water and air dried under shade at room temperature. The dried sample was ground into powder by an analytical mill.

2.2. Apparatuses and chemicals

Apparatuses and instruments utilized during the laboratory work include separator funnel, rotary evaporator, Petri dish, incubator, autoclave, Bench top analytic mill (Tw 135), and UV-Visible Spectrophotometer (DU-8800D). Solvents used for extraction were n-hexane, chloroform and methanol (all were analytical grade solvents). Chemicals like hydrochloric acid, sulphuric acid, Ferric chloride, Mayer’s reagent, ammonia, barium chloride, acetic anhydride, α-naphthol, iodine, KI, and HgCl₂ were used for phytochemical screening. DMSO and Gentamicin were used for an antibacterial test while DPPH and ascorbic acid were utilized for antioxidant evaluation.

2.3. Extraction of plant sample

Dried and powdered root of Clematis hirsuta (250 g) was soaked with n-hexane (1500 mL) for 72 hr with intermittent shaking and then filtered first using cotton plug followed by Whatmann No 1 filter paper. The marc was allowed to dry and then soaked with chloroform (1500 mL) for 72 hrs and filtered. The same procedure was repeated for methanol (1500 mL). The filtrates were pooled and concentrated (separately for each solvent) under reduced pressure. The semisolid mass was made to dry at room temperature, the dried masses were weighed, and the percentage yield was calculated according to Equation (1)(1) $\mathrm{\%}Yield=\frac{Massofcrudeextract}{Totalmassofsampleused}x100$(1) . The dry sample was stored in the refrigerator at 4^OC as described in (Peter et al., 2009) until required for use.

(1) $\mathrm{\%}Yield=\frac{Massofcrudeextract}{Totalmassofsampleused}x100$(1)

2.4. Preliminary phytochemical screening

The collected plant extracts were subjected to qualitative standard screening tests for secondary metabolities such as alkaloids, saponins, tannins, steroids, terpenoids, phenols, flavonoids, glycosides and carbohydrates according to the methods discussed in the literatures (Gul et al., 2017; Harbone, 1973; Natesan et al., 2015; Yadav et al., 2014).

2.5. Antibacterial test

2.5.1. Test organisms

Four bacterial strains (one gram positive): Staphylococcus aureus and three gram negative: Escherichia coli, Salmonella thyphi and Pseudomonas aeruginosa were obtained from Department of biology, microbiology laboratory of Wollega University.

2.5.2. Preparation of test solutions

About 0.01 g of n-hexane, chloroform and methanol crude extracts were dissolved in 10 mL of DMSO separately to test antibacterial activities. Then the resulting concentration was diluted to get the second concentration (1.0 mg/mL, 0.5 mg/mL).

2.5.2.1. Preparation of inoculums

Bacterial strains from the stock cultures were streaked on Mueller Hinton Agar containing the extracts and incubated at 37^OC for 24 hrs. The disc diffusion technique (Baeur et al., 1966) was used to evaluate the antibacterial activity of the plant extracts. Broad-spectrum antibiotic (Gentamicin) was used as positive control and the disk soaked with DMSO as a negative control to compare the result of effects of plant extract. After 24 hrs the antibacterial activity was evaluated by measuring the diameter of the growth inhabited zone based on the method used by Doughari et al. (2007) and compared with the standard antibiotic used.

2.6. DPPH radical scavenging assay

The free radical scavenging ability of the extracts was tested by DPPH radical scavenging assay as described by Blois (1958). The hydrogen atom donating ability of the plant extractives was determined by the decolorization of the methanol solution of DPPH. The violet/purple color of 2,2-diphenyl-1-picrylhydrazyl (DPPH) in methanol solution changes to yellow in the presence of antioxidants.

About 0.01 g of methanol crude extract of Clematis hirsuta was dissolved in 5 mL of methanol to obtain final Stock solution of 2.000 mg/mL and four different concentrations were prepared by serial dilutions (1.000 mg/mL, 0.500 mg/mL, 0.250 mg/mL and 0.125 mg/mL) (Shekhar & Anju, 2017). In DPPH assay 1 mL of 0.1 mM methanol DPPH solution was mixed with 3 mL of different concentration of respective extract. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 minutes followed by taking absorbance measurement at 517 nm on UV-VIS Spectrophotometer. Ascorbic acid was used as a reference. Percentage DPPH radical scavenging activity was calculated using Equation (2)(2) $\mathrm{\%}\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{\left\{\left(Ao-A1\right)\right\}}{Ao}x100$(2) :

(2) $\mathrm{\%}\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{\left\{\left(Ao-A1\right)\right\}}{Ao}x100$(2)

Where A₀ is the absorbance of the control and A₁ is the absorbance of the extractives/standard (Tiwari et al., 2011).

Then % of inhibition was plotted against concentration, and from the graph IC₅₀ was calculated.

3. Results and discussion

3.1. Determination of extraction yield

Extraction yeild was calculated according to the method used by Zhang et al. (2007) and the data is given in Table 1.

Table 1. Percent yeild of n-hexane, chloroform and methanol extract of the root of Clematis hirsuta

Plant extract	Dry powder(g)	Yield(g)	Yeild(%)
n-Hexane extract	250	1.5	0.60%
Chloroform extract	246	3	1.22%
Methanol extract	240	15	6.25%

The percentage yields of plant extraction are mainly dependent on the solvent used in the extraction. Different polarity index solvents give different percentages yield and extract different phytochemical compounds. In agreement with this methanol extract gave the largest mass of phytochemicals compared to that of n-hexane and chloroform extracts. This may be due to the highest polarity index of methanol as compared to other solvents used.

3.2. Phytochemicals screening result

Phytochemical screening test of the crude extracts of the root of Clematis hirsuta showed the presence of alkaloids, saponins, tannins, phenols, flavonoids, glycosides and carbohydrates. While methanol extract gave a positive test for all phytochemicals tested except steroids and terpenoids, n-hexane extract showed the absence of all secondary metabolities tested in the root of Clematis hirsuta (Table 2).

Table 2. Phytochemicals found in the root of Clematis hirsuta crude extract

S. no.	Phytochemical screened	Standard of test	Plant extracts
			n-hexane	Chloroform	Methanol
1	Alkaloids	Mayer’s test	-	-	+
2	Saponins	Foam test	-	-	+
3	Tannins	Braymer’s test	-	+	+
4	Steroids	Salkowski test	-	-	-
5	Terpenoids	Salkowski test	-	-	-
6	Phenols	Ferric chloride test	-	-	+
7	Flavonoids	Lead acetate test	-	-	+
8	Glycosides	Keller kelliani test	-	+	+
9	Carbohydrates	Molisch’s test	-	+	+

‘+’ indicate presence and ‘-’absence of phytochemicals.

Medicinal plants contain biologically active chemical substances (phytochemicals) such as saponins, tannins, essential oils, flavonoids, alkaloids and other chemical compounds, which have preventive and curative properties. These complex chemical substances of different compositions are found as secondary plant metabolites in one or more of these plants and are useful for humanity (Okigbo et al., 2008). Clematis hirsuta, in Ethiopia, has been reported to be used for treating edema, leishmaniasis, herpes, hemorrhoids and tumor on the neck, among others (Teklehaymanot et al., 2007). Powder made from barks, leaves and stems of the plant is applied directly to affected tumor sites. The infusion from leaves of Clematis hirsuta is drunk for the treatment of gonorrhea, syphilis and sore throat. The leaves were also used for the treatment of leprosy, fever and various skin diseases. To clear a stuffy nose, the leaf extracts are sniffed, which causes sneezing. It is also used for headache and the common cold (Pankhurst, 2001). Abdel-Kader and co-workers reported the presence of sterols and triterpenes in petroleum ether and butanol extracts of Clematis hirsuta and demonstrated its anti-inflammatory effect on rat model (Abdel-Kader et al., 2008). Hawaze et al. (2012) reported the presence of proteins, fixed oils, carbohydrates, tannins, saponins, flavonoids, and steroids in the leaf extracts of Clematis longicauda and Clematis burgensis. However, sterols and terpenoids were not detected in the root extract of Clematis hirsuta. Therefore, the reported traditional medicinal value of the plant is most probably associated with those phytochemicals detected in the plant sample.

3.3. Antibacterial activity result

Bacterial growth inhibition data were given in Table 3. The antibacterial activity was determined by measuring the inhibition zone in diameters (mm) and was evaluated according to the parameters suggested by Alves et al. (2000). Methanol crude extract showed a strong inhibition zone (35 mm) at 1.0 mg/mL concentration against E. coli (Table 3, Figure 2). This is promising as compared to the reference gentamicin (26 mm) on the same pathogen. n-Hexane extract did not show any inhibition against all tested bacterial strains. Chloroform extract exhibited moderate inhibition against two of the tested pathogens (S. aureus and P. aeruginosa).

Figure 2. Zone of inhibition produced by solvent crude extracts of root of Clematis hirsuta at 1.0 mg/mL

Table 3. Antibacterial efficacy of extracts against pathogens at 1.0 mg/mL and 0.5 mg/mL concentration

Extract/Bacterial strain		Staphylococcus aureus	Salmonella thyphi	Escherichia coli	Pseudomonas aeruginosa
n-Hexane	1.0 mg/mL	-	-	-	-
	0.5 mg/mL	-	-	-	-
Chloroform	1.0 mg/mL	9 mm	-	-	9 mm
	0.5 mg/mL	-	-	-	-
Methanol	1.0 mg/mL	11 mm	15 mm	35 mm	12 mm
	0.5 mg/mL	10 mm	-	14 mm	10 mm
Gentamicin		28 mm	25 mm	26 mm	25 mm

The root extract of Clematis hirsuta demonstrated the broad-spectrum nature of its constituents. The leaves of Clematis hirsuta were also reported to possess antifungal activity (Cos et al., 2002; Gruenwald et al., 2000); antibacterial activity (Habtamu and Mekonnen, 2017), petroleum ether and butanol extracts of Clematis hirsuta revealed the presence of sterols and triterpenes and has anti-inflammatory on rat model (Abdel-Kader et al., 2008). The study previously undertaken on different species in this genus has been indicated that different parts of the plant have different antimicrobial activities. C. papuasica leaves and stem bark showed a wide spectrum of antibacterial activity (Khan et al., 2001), the leaf extract of C. simensis was reported to possess antibacterial activity (Desta, 1993), phytochemicals like triterpenoid, saponins and alkaloids isolated from different parts of the species belonging to the genus are responsible for cytotoxic (Yan et al., 2009), antibacterial (Ding et al., 2009), and antifungal (Chen et al., 2009) activities. The methanol and petroleum ether extracts of the leaves of Clematis longicauda and Clematis burgensis showed antibacterial and antifungal activity in a concentration-dependent manner (Hawaze et al., 2012).

The observed bioactivities of the root extracts of Clematis hirsuta might be due to the presence of secondary metabolities like tannins, flavonoids and saponins. Tanins and flavonoids are known to exhibit antimicrobial, anti-inflammatory, analgesic and antioxidant properties (Edewor & Usman, 2012). Tannins precipitate microbial protein rendering it unavailable to bacteria (Banso, 2009). Tannin-containing plants are for treating wounds and intestinal disorders such as diarrhea and dysentery, thus exhibiting antibacterial activity (Abubakar, 2009). Saponins are also reported to have antibacterial activity (Maatalah et al., 2012).

3.4. Antioxidant activity of methanol crude extract

Antioxidant is a molecule that inhibits the oxidation of other molecules by accepting an electron or free radicals produced during oxidation. Unless trapped by antioxidants, these free radicals can start chain reactions in a cell that can cause damage or death to the cell (Ames et al., 1993, Shenoy & Shirwaiker, 2002).

The antioxidant activity of the methanol crude extract and its serially diluted solutions of the root of Clematis hirsuta was tested for their antioxidant activity using DPPH radical scavenging assay (Table 4). DPPH radical is a stable organic free radical with an absorption band at 517 nm. It loses this absorption on accepting an electron donated by an antioxidant compound or a free radical species, which results in a visually noticeable color change from purple/deep violet to yellow and this can be quantitatively measured from the changes in absorbance. It can accommodate many samples in a short period and is sensitive enough to detect active ingredients at low concentrations (Hseu et al., 2008).

Table 4. The DPPH free radical scavenging activity of the root extract of Clematis hirsuta

Concen tration (mg/mL)	Root of Clematis hirsuta			Ascorbic acid
	Absor bance	% DPPH inhibition	IC50	Absor bance	% DPPH inhibition	IC50
0.125	0.7550	28.04	0.599	0.5400	48.53	0.570
0.250	0.5916	43.61		0.3134	70.13
0.500	0.5816	44.57		0.1991	81.02
1.000	0.3519	66.46		0.0842	91.97
2.000	0.0181	98.27		0.0432	95.88

In this study, it was observed that methanol crude extract of the root of Clematis hirsuta had radical scavenging activities in a concentration-dependent manner. Absorbance increased as concentration decreased from 2.000 mg/mL to 0.125 mg/mL indicating higher free radical scavenging activities of samples indicated by lower absorbance at 517 nm (Princen et al., 1992). The root extract of Clematis hirsuta exhibited strong radical scavenging activity with a percentage DPPH inhibition of (98.2%) at a concentration of 2.000 mg/mL as compared to the reference ascorbic acid (95.8%) at the same concentration. However, at lower concentrations the %DPPH inhibition of the root extract was lower than that of ascorbic acid with similar concentrations (Figure 3).

Figure 3. Bar graph of %DPPH inhibition of methanol extract of root of Clematis hirsuta and Ascorbic Acid

IC₅₀value gives effective concentration required for 50% inhibition (Fridovich, 1995). The methanol extract reduced DPPH radicals significantly as it is evident from its low IC50 (0.59 mg/mL) value. At the same concentration, the IC50 value of the standard ascorbic acid is (0.57 mg/mL) (Table 4).

Free radicals generated in our body are assumed to be the cause for diseases like inflammation, atherosclerosis, stroke, heart diseases, diabetes mellitus, multiple sclerosis, cancer, Parkinson’s disease, and Alzheimer’s disease (Doughari et al., 2009; Rahman et al., 2012). Plants constitute an important source of active natural products, especially polyphenols (such as flavonoids, phenyl propanoids, Phenolic acids, tannins, etc.) that can inhibit or at least suppress the harmful effect of these free radicals. Almost all polyphenols share the same chemical patterns (Phenolic group) that help them to react as hydrogen donors and hence neutralize free radicals (Riaz et al., 2012). The radical scavenging properties exhibited by the root extracts of Clematis hirsuta might be due to the presence of phytochemicals like flavonoids, phenols, tannins and saponins.

4. Conclusions

Medicinal plants contain active ingredients to cure disease. The therapeutic properties of plants could be due to their antioxidant and antimicrobial effects of the secondary metabolities in them. The phytochemical screening test of crude extract of Clematis hirsuta confirmed the presence of phytochemicals such as alkaloid, saponins, flavonoids, phenols, tannins, glycosides and carbohydrates. Methanol extract of the root of Clematis hirsuta showed moderate bacterial growth inhibition as well as antioxidant activity. The observed antibacterial and antioxidant activities support the traditional use of this plant for the treatment of various ailments. Therefore, Clematis hirsuta root can be a potential source in search of potent natural antioxidants.

Acknowledgments

The authors are grateful for Wollega University.

Additional information

Funding

The authors received no direct funding for this research.

Notes on contributors

Zelalem Abdisa

Zelalem Abdisa completed his First Degree in Applied Chemistry, Master’s Degree in Organic Chemistry, and his PhD in Organic Chemistry all from Addis Ababa University, Ethiopia. He has been teaching at Undergraduate and Graduate programs at Wollega University for the last 13 years. He had also been working as department head, vice dean, and director at Wollega University. He is currently working at the position of associate dean for postgraduate research at Wollega University. He is now an assistant professor in Organic Chemistry.