Comparison of phytochemical extraction solvents for Andrographis paniculata

Navjot Kaur, Jeena Gupta*

Department of Bioengineering and Biosciences, Lovely Professional University (LPU),

Phagwara, Punjab India - 144411

*Corresponding Author E-mail: jeena.20104@yahoo.co.in

ABSTRACT:

Andrographis paniculata is a medicinal plant known to possess many health benefits like antioxidant, antibacterial, antitumor, antidiabetic, antithrombotic, anti-inflammatory and antiviral activities. In spite of all known beneficial effects, there is no standardized protocol for extraction of bioactive components from this plant. In the present study we compare the different extraction solvents (acetone, chloroform, distilled water, ethyl acetate, hexane and methanol) for their efficiency in extracting Andrographis Paniculata plant bioactive components and to evaluate the plant regeneration protocol under laboratory controlled conditions using different hormone combinations. Our results shows that chloroform extract shows the maximum phenol concentration (10.6 ±0.3 mg of Gallic acid equivalent/ 100 g of extract) and maximum antioxidant activity (47.75± 0.008 percent inhibition), however lack in the extraction of saponins and tannins. Using hexane as an extraction solvent results in the maximum extraction of flavonoids with 9.2 ± 0.4 mg of quercetin equivalent/ 100 g of extract but reduced phenol concentration (0.8 ± 0.01 mg of Gallic acid equivalent/ 100 g of extract) and antioxidant activity (13.1 ± 0.01% inhibition). To best of our knowledge this is the first report which directly compares the phytochemical extraction solvents for Andrographis paniculata and shows that different solvents are having different phytochemical extraction potential for this plant.

KEYWORDS: Andrographis paniculata, Phytochemical, Extraction solvent, chloroform, Hexane and DPPH

INTRODUCTION:

Andrographis paniculata is a small annual herb with a strong bitter taste and belongs to the family Acanthacea. It is found in tropical and southeast Asia and is commonly called ‘king of bitter’ due to its extremely bitter taste¹. Other common names of this plant are “Hempedubumi” in Malaysia, “Chuan Xin Lian” in China, “Fa Thalai Chon” in Thailand, “Senshiren” in Japan and “Kalmegh” in India². This plant is known to contain many bioactive compounds like steroids, phenols, terpenoids, alkaloids, saponins and flavonoids, primarily used by the plant for protection against pathogens³. Many medicinal properties accounts for these bioactive components for which it is used as an ayurvedic medicine since long times (Table 1).

Table 1: The basic structures and medicinal properties of various phytochemicals present in Andrographis paniculata plant

Phytochemical	Structure	Medicinal properties
Terpenoids	Isoprene units (consisting of five-carbons with two unsaturated bonds and a branched chain) in which the methyl groups are replaced by oxygen atoms	Anticarcinogenic, antiulcer, antimicrobial, antioxidant, antimalarial and antidiuretic ²²
Phenols	A aromatic ring with a hydroxyl group	Antimicrobial, antioxidant ²³
Flavonoids	Phenolic aromatic molecules having a carbonyl group	Anti-inflammatory, antithrombotic, antioxidant, antiviral ³
Saponins	One or more hydrophilic glycoside units with lipophilic triterpene derivative	Antidiarrheal, anticancer, and antihelmintic ²⁴
Tannins	One or more hydrophilic glycoside units with lipophilic triterpene derivative	Antioxidant, antiperoxidative, antimicrobial, antimutagenic, antidiabetic and antiviral ²⁴

The major bioactive compound found in high amounts in Andrographis paniculata is Andrographolide, which is a diterpenoid lactone and found particularly in leaves. Primarily used by the plant for protection, these compounds possess many health benefits like antioxidant⁴, antibacterial^5,6, antitumor, antidiabetic, antithrombotic, anti-inflammatory⁷and antiviral activities⁸. Andrographis paniculata is used in Traditional Chinese medicine (TCM) since ancient times to release body heat and toxins. It is used as an oral remedy against common cold, dysentery, fever, tonsillitis, diarrhoea, liver diseases, inflammation, herpes, influenza, sinusitis, antherosclerosis, insect and snake bites. It is also used for the treatment of leprosy, gonorrhea, scabies, boils, skin eruptions due to its blood purifying properties⁹. The ethanolic extract of Andrographis paniculata was also found to be antifungal¹⁰. Further the report had demonstrated by Brine shrimp lethal test that Andrographis paniculata plant extract is not cytotoxic¹¹. Even many Siddha formulations (a traditional medicine system of south India) were found to contain good content of andrographolide¹².

This plant is normally found in wild fields; however, it is difficult to meet the commercial demand due to limitations in the conventional production system for this plant ¹³. In vitro propagation is an alternative and efficient method to obtain the maximum yield of this valuable plant derived pharmaceuticals. Past research efforts for the regeneration of Andrographis paniculata have been taken by various groups using different plant parts like Karuppusamyet et al., in 2010 regenerated 30 day old seedlings¹⁴ and Bidari S.K. et al., in 2012 regenerated nodal explants of the plant by using different hormone combination ¹⁵. However more research efforts are needed to provide a standard regeneration protocol.

Although the medicinal properties of this plant Andrographis paniculata are well known since ancient time, however, there is no standardized protocol for the extraction of active components from this plant. So in view of this the present study was undertaken to compare the efficiency of different extraction solvents in extracting the various phytochemicals present in the plant. We further also want to standardize its regeneration protocol under laboratory controlled conditions. We here provide the first report which shows that chloroform is the best extraction solvent for the extraction of the phytochemicals from the dried leaves of Andrographis paniculata.

EXPERIMENTAL:

Materials:

In this study, all the chemicals were provided by Hi-Media Co. including methanol, acetone, chloroform, ethyl acetate and hexane.

Collection of the plant sample and extract preparation:

The plant was taken from the Herbal Garden of Lovely Professional University. The plant authentication for Andrographis paniculata was done by expert botanist from Department of Botany, Lovely Professional University. The plant leaves were shade dried and crushed to make the fine paste. Extraction with different solvents like acetone, chloroform, distilled water, ethyl acetate, hexane and methanol have been done in soxhlet apparatus. Briefly, for every 200 ml of the each solvent, 25g of the crushed plant leaves powder was used for the soxhlet extraction. After extraction for 3 consecutive days, the extracts were placed in water bath at 55 ̊ C for evaporation and thus the crude extract of the plant for the each solvent was obtained.

Phytochemical Screening:

Biochemical tests have been done to check the presence of different phytochemical such as alkaloids, flavonoids, saponins, steroids and tannins in the above mentioned Andrographis paniculata plant extract by the following procedure:

Test for alkaloids:

10 mg of the each extract was taken and was dissolved in 2 ml of the Wagner’s reagent for different extracts. After dissolving the both, the formation of reddish brown colored precipitates confirms the presence of alkaloids in the plant extract.

Test for flavinoids:

10 mg of the each extract was taken and the few drops of diluted NaOH was added to the each extract. The appearance of yellow color which disappears or become colorless on adding few drops of diluted H₂SO₄ confirms the presence of flavonoids in the plant extract.

Test for saponins:

10 mg of the each extract was taken and each extract was diluted with 20 ml of distilled water. The test tube was then shaken for 15 minutes by hand and the formation of foam on top of the test tube shows the presence of saponins in the plant extract.

Test for steroids:

10 mg of the each extract was taken and the 1 ml of concentrated H₂SO₄ had been added to the each extract by the side walls of the test tube. Appearance of dark reddish green color confirms the presence of steroids in the plant extract.

Test for tannins:

10 mg of the each extract was taken and dissolved in 45% of the ethanol. Then the test tube was boiled for 5 minutes and 1 ml of 15% ferric chloride solution was added to each. The appearance from greenish to black color confirms the presence of tannins in the plant extract.

Antioxidant activity:-

Blois method ¹⁶ was used to determine the free radical scavenging activity of the plant extract by using DPPH (2,2-diphenyl-1-picrylhydrazyl). Briefly, 0.2 mM DPPH solution was made using methanol. Ascorbic acid was taken as the standard. 10 µg of the each extract was dissolved in 2ml of the mother solvent and 1 ml of prepared DPPH was added in all the tubes. The tubes were then kept in the dark for 60 minutes and the absorbance of all the samples was taken at 517 nm using spectrophotometer.

The % inhibition was calculated by using the formula =

[(A_control-A_extract) / (A_control)] * 100

Total phenolic content in the plant:-

To determine the total phenolic content of the plant, gallic acid was used as the standard. 20 µl of the each extract was taken and volume adjusted to 2 ml using parent solvent. 200 µl of FCR and 500 µl of 20 % Na₂CO₃have been added to each tube. Then the reaction mixture was incubated for 1 hour at room temperature and the absorbance was measured at 760 nm.

Quantification of the flavonoids in the plant:-

For the quantification of the flavonoids, quercetin was used as the standard. Different concentrations of the quercetin (200 µl, 400 µl, 600 µl, 800 µl & 1 ml) have been used to make standard curve. For test samples 10 µl of each extract was dissolved in 100 µl of particular parent solvent. The final volume for the each sample was adjusted to 2 ml by adding 100 µl of potassium acetate, 100 µl of aluminium chloride and rest distilled water. The samples were then incubated for 30 minutes at the room temperature and the O.D. was taken at 470 nm.

Statistical analysis

Experimental values are expressed as mean ± SEM. Comparison of mean values between various groups was performed by one way-analysis of variance (one way- ANOVA). P-value < 0.05 was considered to be significant.

RESULTS AND DISCUSSION:

Extraction of active components from Andrographis paniculata plant using different solvents

The extracts from the dried leaves of the plant were made by using different solvents: acetone, chloroform, distilled water, ethyl acetate, hexane and methanol for three consecutive days each in soxhlet apparatus. The extracted aqueous extracts were kept in the water bath at 55 ̊ C for evaporation of the solvent and the crude extract of the plant for the each solvent was obtained. After obtaining the extracts, the percentage yield of the extracts was calculated (Table2). Our results show the maximum percent yield in obtained by using acetone as an extraction solvent (88.2%), followed by distilled water (36.76%), chloroform (27.16%), ethyl acetate (24.4%), methanol (21.32%) and hexane (17.88%). Hexane extraction results in minimum percent yield.

Table 2: Percentage yield of the extracts made from Andrographis paniculata plant using different extraction solvents

EXTRACT NAME	PERCENT YIELD OF EXTRACT	COLOUR OF EXTRACT
Acetone	88.2%	Dark green
Chloroform	27.16%	Light green
Distilled water	36.76%	Dark brown
Ethyl acetate	24.4%	Dark green
Hexane	17.88%	Light green
Methanol	21.32%	Dark green

(Weight of extract obtained / Weight of dry powder used) * 100

Phytochemical screening:-

Phytochemical screening tests have been performed to detect the presence of bioactive components in the plants. The results for the tests for the presence of saponins, tannins, alkaloids, steroids and flavonoids were as follows (Table 3).

· Test for saponins:- The extracts of all the solvents were diluted with the distilled water and the test tubes were shaken for 15 minutes by hand and the formation of the foam on the upper layer of the test tube showed the presence of saponins in it. All the extracts of the different solvents, except chloroform showed the positive result for the presence of saponins in the plant. The tests were performed in triplicates.

· Test for flavonoids:- All the extracts of the different solvents showed the positive result for the presence of flavonoids in the plant due to the appearance of the yellow color in all.

· Test for alkaloids:- The extracts of the different solvents showed the appearance of reddish brown color in the test tubes which confirms the presence of alkaloids in the plant extracts obtained using different solvents.

· Test for steroids:- All the extracts of the different solvents showed the appearance of dark reddish green colour in the test tubes which confirms the presence of steroids in all the plant extracts.

· Test for tannins:- All the extracts of the different solvents, excluding chloroform showed the positive result for the presence of tannins in the plant due to the appearence of greenish black colour in the test tubes. The chloroform extract showed the negative result for the presence of tannins may be due to less solubility.

Antioxidant activity

An antioxidant activity of all the plant extracts was observed using DPPH (2,2-diphenyl-1-picrylhydrazyl) and ascorbic acid as the standard. A standard curve was made and DPPH activity was observed by measuring OD at 517 nm. We observed that extracts from Andrographis paniculata plant formed using chloroform, distilled water and ethyl acetate shows the maximum antioxidant activity with 47.75± 0.008 %, 42.9 ± 0.009 % and 45.95 ± 0.0015 % inhibition respectively followed by acetone (35.75 ± 0.03) and methanol (31.85 ± 0.0025). Whereas hexane extract shows the least antioxidant activity with only 13.1 ± 0.01% inhibition (Table4).

Total flavonoid quantification in the plant:-

Total flavonoid concentration was quantified by using quercetin as the standard and measuring OD at 470 nm. Using hexane and methanol as an extraction solvent results in the maximum flavonoid extraction with 9.2 ± 0.4 and 6.5 ± 0.1mg of quercetin equivalent/ 100 g of extract respectively, followed by distilled water (6.1 ± 0.9 mg of quercetin equivalent/ 100 g of extract), acetone (5.6 ± 0.2 mg of quercetin equivalent/ 100 g of extract) and chloroform (5.6 ± 1 mg of quercetin equivalent/ 100 g of extract). When we use ethyl acetate as an extraction solvent, it results in the least extraction of flavonoids with 3.2 ± 0.4 mg of quercetin equivalent/ 100 g of extract from Andrographis paniculata plant leaves (Table4).

Total phenolic content in the plant:-

Total phenolic content in the plant was estimated using Gallic acid as a standard. The OD was taken at 760 nm. Maximum phenol concentration was observed when chloroform (10.6 ±0.3 mg of Gallic acid equivalent/ 100 g of extract) and distilled water (4.7 ± 0.7 mg of Gallic acid equivalent/ 100 g of extract) was used as extraction solvent followed by acetone (3.4 ± 0.2 mg of Gallic acid equivalent/ 100 g of extract) and ethyl acetate (2.45 ± 0.3 mg of Gallic acid equivalent/ 100 g of extract). When we use hexane and methanol as extraction solvent, it results in the least extraction of flavonoids from Andrographis paniculata plant leaves with 0.8 ± 0.01 and 0.22 ± 0.06 mg of Gallic acid equivalent/ 100 g of extract (Figure1 and Table4). This data directly correlates with antioxidant activity shown by these plant extracts (Figure1 and Table4).

DISCUSSION:

Andrographis paniculata is a potent detoxifier and has been used in traditional systems since centuries for the treatment of many diseases particularly infections, fevers and gastric problems¹⁷. Being enriched in many active phytochemicals, numerous scientific reports have evaluated the pharmacological properties of this plant like anti-diabetic, anti-inflammatory, anticancer, immunomodulation, antipyretic, antibacterial, and anticarcinogenic ^18-20. However, present study was conducted with an objective to identify the best extraction solvent which can be used to extract the maximum amount of the phytochemicals from the shade dried Andrographis paniculata plant leaves.

Qualitative biochemical estimations were conducted to detect the presence of different phytochemicals in the dried Andrographis paniculata plant leave’s extracts obtained by using different solvents i.e. methanol. acetone, hexane, ethyl acetate, chloroform and distilled water. Our results highlights that all the extracts formed by using different solvents from Andrographis paniculata plant leaves were enriched in phytochemicals like saponins, flavonoids, alkaloids, steroids and tannins. However, saponins and tannins were found to be absent in the extract made by using chloroform. It may be due to poor solubility of these phytochemicals in chloroform (Table 2).

We have further conducted a quantitative study to detect the amount of the total flavonoids and total phenols in these plant extracts for direct comparison and have demonstrated that hexane extract contains the maximum amount of flavonoids and chloroform extract is enriched in phenols. Previous study had also demonstrated the isolation of different flavones from roots and stem of Andrographis paniculata plant using hexane, methanol and chloroform as extraction solvent²¹. But our results shows that different solvent are having different abilities in efficiently isolating various phytochemicals from dried leaves of Andrographis paniculata plant.

CONCLUSION:

Andrographis paniculata plant extract commonly called “Kalmegh” extract is known to possess many beneficial effects and is used since traditional times for the treatment of various diseases. In the present study we perform a direct comparison between the different extraction solvents for their efficiency in extracting the phytochemicals from Andrographis paniculata dried leaves. Our results clearly demonstrate that chloroform is the best extraction solvent for phenols and possess maximum antioxidant activity whereas hexane results in maximum yield of flavonoids. To best of our knowledge this is the first study which directly compares the six extraction solvents for their potential to extract the various phytochemical from Andrographis paniculata plant and shows that different solvents are efficient in extracting different phytochemicals.

AUTHOR’S CONTRIBUTIONS:

Navjot Kaur conducted all the experiments, compiled results with statistical analysis and wrote manuscript. Jeena Gupta planned and monitored all the experiments and formatted the manuscript.

CONFLICT OF INTEREST STATEMENT:

None declared.

ACKNOWLEDGMENT:

This work was supported by a grant from Lovely Professional University (LPU).

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TEST	METHANOL	EHTYL ACETATE	CHLOROFORM	DISTILLED WATER	ACETONE	CONTROL
ALKALOIDS	++	+	+++	+	+	-
FLAVONOIDS	++	+	+	+++	++	-
SAPONINS	-	++	+	+++	++	-
STEROIDS	++	++	+	+	++	-
TANNINS	++	+	-	++	++	-

EXTARCT	FLAVONOID CONC. (mg of Quercetin equivalent/ 100 g of extract)	PHENOL CONC. (mg of Gallic acid equivalent/ 100 g of extract)	% INHIBITION ACTIVITY OF DPPH (%)
ACETONE	5.6 ± 0.2	3.4 ± 0.2***	35.75 ± 0.03***
CHLOROFORM	5.6 ± 1	10.6 ± 0.3***	47.75 ± 0.008***
DISTILLED WATER	6.1 ± 0.9	4.7 ± 0.7***	42.9 ± 0.009***
ETHYL ACETATE	3.2 ± 0.4	2.45 ± 0.3***	45.95 ± 0.0015*
HEXANE	9.2 ± 0.4	0.8 ± 0.01**	13.1 ± 0.01
METHANOL	6.5 ± 0.1	0.22 ± 0.06	31.85 ± 0.0025