A Preliminary Study on Phytochemical Screening, Proximate Analysis and Anti-Bacterial Activities of Andrographis paniculata Seed Extract

 

Geetha S1,  Rajeswari S2,

1Associate Professor, Department of Biochemistry, School of Life Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Chennai, Tamil Nadu, India.

 2Ph.D scholar, Department of Biochemistry, School of Life Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Chennai, Tamil Nadu, India.

*Corresponding Author E-mail: geethasubramaniam05@gmail.com

 

ABSTRACT:

A preliminary investigation was done to screen the phytochemical contents and to find out the proximate composition and antibacterial activity of Andrographis paniculata seeds. Standard analytical procedures were followed and four different solvent extractions were done. Qualitative phytochemical screening of Andrographis paniculata seeds showed the presence of important phytochemical compounds in methanol seed extract viz., Flavonoids, Saponins, Tannins, Phenolic compounds, Terpenoids and Glycosides. Chloroform, ethyl acetate and methanol extracts reported the presence of tannins, Phenols and terpenoids. Flavonoids were present in chloroform and methanol extracts. The hexane extract did not show positive for any of the tests. Alkaloids was found to be absent in all the solvent extracts. Further, tannin concentration was found to be in high in methanol extract in comparison to other solvent extracts. Proximate composition recorded methanol extractive value of about 57.75% and was higher than water extractive value (46.23%). The moisture content of the crude drug was found to be 21.40% and foaming index was < 100%. Antibacterial activity was studied by assessing the minimum inhibitory concentration of methanol extract for bacterial strains. The MIC values of Salmonella typhi 59, Vibrio cholera and Shigellaboydii 8 was found to be 100 µg/ml and for Bacillus licheniformis and  Staphylococcus aureus, MIC was found to be 10µg/ml. The preliminary investigation suggests that Andrographis paniculata seed extract have a rich profile of phytochemicals with significant proximate composition and good antibacterial activity. The findings can be taken as an initiative step for finding out the promising agents responsible for its phytochemical and antibacterial activities. This could further pave way to the research with respect to antioxidant studies of Andrographispaniculata seeds.

 

KEYWORDS: Andrographis paniculata seed extract, Phytochemicals, Antibacterial, Antioxidant, MIC (Minimum Inhibitory Concentration).

 

 


 

 

 

 

 

 

 

 

 

 

 

INTRODUCTION:

In recent years, interest in medicinal plants has grown enormously as an alternative to allopathic medication, globally, owing to the increased resistance of microorganisms and adverse side effects of synthetic drugs. The issue has paved way for numerous scientific investigations and evaluation of plants for their biological and pharmacological effects on human beings. At present, more focus is given on herbal products to supplement the diet and immune boosters, for better immunity, prevention of infections and diseases for all age communities. Discovery of new herbal remedies is the need of the hour to meet the future challenges in health concerned issues in the near future.

Andrographis paniculata (Burm.F) Nees, commonly termed as “king of bitters,” is an annual, branched, herbaceous plant of Acanthaceae family. It is largely distributed in tropical Asian countries and subtropical region of South East Asia, Malaysia, Thailand and  Indonesia. The plant is best grown in open or slightly shaded area with an easy access of water and reaches the height of 30 -110 cm with quadrangular green stem.  Leaves appear simple, opposite and lanceolate with a length of 2-12 cm and 1-3 cm wide. The flowers possess calyx with small and linear 5 sepals.  The seeds are very small, sub quadratet. Native population of the plants are found in Srilanka and all over southern India, where it is termed as Nilavembu, which indicates the centre of origin and diversity of the species1and as Kalmegh in Ayurvedic system.

 

Andrographolide (C20H30O5; MW 350.44), a bicyclic diterpenoid lactone, is the active and major constituent of the leaves and aerial parts of the plant, which possess wide spectrum of pharmacological activities2. The Andrographolide possess antioxidant activity, and inhibits neutrophil production and macrophage migration. Potent Hepatoprotective activity is reported which acts by increasing the bile flow and bile salt production in comparison with Silymarin A3.

 

Many medicinal properties of A. Paniculate have been documented. Significant applications were reported in treating respiratory and intestinal tract infections, sore throat, fever, herpes and other chronic and infectious diseases4. Recently anti dengue activity of bioactive component, andrographolide was reported against the primary dengue vector Aedesaegypti5. Aqueous extract of A. paniculate was found to possess antibacterial activity against both gram positive, gram-negative microorganisms and against many tested pathogenic clinical strains of bacteria6.

 

It was found to possess antioxidant activity, anti-malarial anti-inflammatory, antiparasitic, antispasmodic, anti-diabetic, antidiarrheal, nematicidal, protozoacidal and anticancer activities7.

 

Througout the world, A. paniculata have found its place in siddha, ayurvedic, unani and chinese traditional systems of medicines8. In Tamil Nadu, southern state of India it is called as Nilavembu or Siriyanangai and acts as an Analgesic, antipyretic and anti-inflammatory agents. Nilavembu kudineer was recommended for usage in Ayurveda, siddha and in unani systems of medicine for the treatment of diabetes, malaria, liver cancer and various infectious diseases.

 

 

 

It is mentioned in Indian Pharmacopoeia and is a prominent factor in several ayurvedic formulations. Some of the herbal products with A. paniculata as a major ingredient available in the market viz., Liv 52, A and rographis 60V caps, andrographis tablets (200, 300 and 400mg), Panchanga Churana, Kalmegharasa, Kalmeghnavayaslauh, kalpataruras, etc., at national and international levels.

 

The demand of A. paniculataas a bioremedies increasing day by day due to its importance in the treatment of different ailments. Many literatures are available on various biological activities of A. paniculate leaves, roots, whole plant, but no studies are reported on its seeds. Hence, in the present study, the potential of A. paniculata seeds was evaluated by screening its phytochemical activity, proximate composition and antibacterial activities. The present investigation holds importance that the current study is of first of its kind as so far none of activities have been reported on A.paniculata seeds.

 

MATERIALS AND METHODS:

Collection of Plant Material:

The seeds were obtained from MG NATURALS shop, Oragadam, Chennai, India. The seed samples are wild varieties, which are not micro or macro propagated by humans. The seeds were identified and authenticated by Dr. P. Jayaraman, Director, Institute of Herbal Botany, Plant Anatomy research Centre, Tambaram, Chennai, India (Ref. No. PARC/2018/3694 dated 16.03.2018).

 

Organoleptic Properties:

The organoleptic properties of A. paniculata seeds are evaluated based upon the morphological and sensory profile of the seeds.

 

Preparation and Processing of A. paniculata seeds:

The seeds were cleaned, removed dirt and shade dried for a week’s time. The dried seeds were ground to powder in a blender and stored in a container. A.paniculata seeds are subjected to soxhlet extraction in sequential manner to out to extract the phytochemical compounds. The solvents were used were hexane, chloroform, ethyl acetate and methanol, with increasing polarity index.

 

About 20g of powdered seeds was taken with 250 ml of the solvent and subjected to soxhlet extraction for 4 hours at 65°C25. The extracts obtained were concentrated by vacuum rotary evaporator at 40° C and stored for further use. The percentage yield was calculated by the using the formula,

                           Weight of the dry concentrated crude extract

Yield of   =  --------------------------------------------------------------× 100
extract (%)            Weight of the powdered seed sample

 

Proximate analysis:

The analysis of proximate composition of A. paniculata seeds was carried out to find out the ash content, extractive value, moisture content, loss on drying and foaming index of the seeds by following the standardized methods of Kokate9.

 

Phytochemical evaluation of seed extracts of Andrographispaniculata in different solvents:

A Preliminary Phytochemical screening was carried out with different solvent extracts of A. Paniculata seeds and the analysis was performed according to the standard methods described by Kokate (2000). The tests were assessed depending upon the visual observation of colour change of a specific precipitate after the addition of specific reagents. In addition, tannin concentration in different solvents was also assessed.

 

Evaluation of Antibacterial activity:

The antibacterial activity was determined by broth dilution method and was assessed by measuring the MIC (Minimum Inhibitory Concentration) values. The test was carried out based on the document M07-A9 guidelines.

 

Test Organisms:

The microbial strains used for testing the antimicrobial activity of A.paniculata included two positive bacteria (Bacillus licheniformis (ATCC 10341), Staphylococcus aureus (ATCC ML-59) and three gram negative bacteria (Salmonella typhi (ATCC 59), Vibrio cholera (ATCC 811) and Shigellaboydii (ATCC 8).

 

The seeds of A.paniculata were powdered and subjected to soxhlet extraction. About 200g of the powder was percolated in 1600ml of ethanol and concentrated using rotary evaporator. The extract was stored in sterile dark vials at 4°C, until further use. To determine minimum inhibitory concentration (MIC), broth micro dilution testing was carried out based on document M07-A9for methanol seed extract of A.paniculata. Different concentrations of test extract ranging from 10 to 200 µg/ml extract was taken for assessing its antimicrobial activity. By means of a loop, the colonies were transferred to tryptic soy broth and incubated at 35–37 °C for 24 hours, until the growth matches the turbidity equal to or greater than that of a 0.5 McFarland standard. Sterile distilled water was used to adjust the culture to measure the turbidity equivalent to the McFarland 0.5 standard.

 

MIC were determined visually with the assistance of a reading mirror, as per CLSI (Clinical and Laboratory Standards Institute) guidelines. The growth in each tube or well was compared with that in the positive growth control.

 

RESULTS AND DISCUSSION:

Significant pharmacological activities of theaerial and root parts of A. paniculata was reported in many studies, whereas none of the reports have been documented on A.paniculata seeds. Hence a preliminary investigation was performed on phytochemical, proximal composition and antibacterial properties of A. paniculata seeds. The results were presented as follows. Organoleptic properties are considered to be very important for the evaluation of quality of a drug. The dried and powdered seed were used for evaluation and the results are given in Table.1

 

Table 1: Evaluation of Organoleptic properties

S. No

Organoleptic property

Observation

1

Appearance

Coarse powder

2

Colour

Green

3

Foreign matter

<1%

4

Sand

Absent

5

Odour

Characteristic odour

6

Taste

Intensely bitter

7

Insect Infestation

Nil

8

Rodent contamination

Nil

9

Powder pressed between two filter papers (24 hours)

Stained

 

Table.1 presents the result of organoleptic properties of the A.paniculata seeds.

 

The characteristic odour and the bitter taste could be attributed to the high phenolic content, along with considerable tannin content as reported in the study. The finding is in conformity with previous works of Silvia et al.10, who stated that there exists a positive correlation between total phenolic content and bitter intensity in virgin olive oil. Further, there was no insect infestation and rodent contamination reported in the stored seeds, which could be due to the presence of phenolic content present in the seeds. The result was in agreement with the review work of Showket et al11, who reported the role of plant phenols and polyphenols, their interactions and resistance against insects and pest infestations.

 

Thus the organoleptic evaluation reveals that the seeds possess good insect and rodent repellent properties, as characterized by their odour and bitter taste, which could be due to the presence of phenolic compounds and which may attribute to its various bio activities.

 

Percentage yield of sequential extraction:

As per the findings of Abubacker12, sequential extraction performed by using increasing polarity index of solvents results in the extraction of secondary metabolites from plants. Basri et al.13, have documented that the yield percentage of extraction denotes the ability of a solvent to extract active compounds from plants and depends solely upon the type of solvent used for extraction. The percentage yield of extraction was depicted in the Fig.1. The methanol extraction reported the highest yield of extraction, 12.59±0.19%, followed by aqueous extract with 11.49±0.10%, chloroform with 3.74±0.12% and the lowest percentage yield was reported by Ethyl acetate (2.69±0.15) and hexane (1.57±0.10). The extraction results denotes that the A. paniculata seed extract contains more polar compounds, as it was extracted equally good in alcoholic as well as aqueous extract.

 

 

Fig.1 Percentage yield of A. paniculata seed extract with different polarity index of solvents. Values are expressed as mean±standard deviation (n=3).

 

Proximate Analysis in methanol seed extract of A.paniculata:

Table. 2 shows the proximate compositions of the seed extract of A. paniculata. The results showed the presence of Total ash, Water insoluble ash, Acid insoluble ash and sulphated ash content, its moisture content, methanol and watersoluble extractive values, Foaming and loss on drying values.

 

Table.2 Proximate Analysis of A. paniculata seeds

S. No

Parameters determined

Values in (%) w/w

1

Total Ash

62.25±0.10

2

Water Insoluble ash

58.168±0.11

3

Acid Insoluble ash

22.514±0.24

4

Sulphated ash

28.71±0.11

5

Total moisture content

31.40±0.17

5

Methanol soluble extractive

57.75±0.14

6

Water soluble extractive

55.23±0.10

8

Foaming index

<100

9

Loss on drying

10.95±0.07

Table.2 presents the result of proximate analysis of A. paniculata seeds. Values are expressed as mean± SEM (n=3).

 

 

 

 

The proximate analysis aids in the detection of bioactive dietary elements that could be responsible for the therapeutic properties of the seeds. The high ash content reflects the content of inorganic matter present in the seed extract and is also an indicative of high digestibility of the seed extract. Vidita et al.,14 in their work had mentioned about the significance of ash value in determining the authenticity and purity of a sample and important for qualitative standards.

 

The percentage of results clearly indicates that the seeds stands best as a drug and with associated effects. The methanol extractive value (57.75%) followed by water extractive value (55.23%). Moisture is one of the major factor that accounts for stability of the drugs and formulations. The moisture content of the crude drug was found to be 21.40%. The low moisture content may show high stability for the drug. The foaming index denotes the ability of an aqueous decoction of seed extract to produce foam. The result being recorded as < 100% denotes that the value is non- significant. This further suggest that in future, drug formulation will not affected by foaming issues.

 

Loss on drying determines both the water and volatile matter in the crude drug. The value recorded was found to be 10.95%, which denotes that the seed extract upon formulation as drug would not be subjected to excess dryness.

 

Qualitative phytochemical Screening:

 Phytochemicals are the metabolites produced by plants that imparts characteristic color, flavor, smell and texture to the plants. Phytochemicals were reported to possess various pharmacological and antimicrobial activities15. The results of qualitative analysis of phytochemicals were presented in Table.3. The result has revealed the presence of Flavonoids, Alkaloids, Saponins, Tannins, Phenolic compounds, Terpenoids and Glycosides. Phenolic compounds, terpenoids and tannins were present in all extracts expect hexane, whereas glycosides were present in ethyl acetate and methanol extracts. Presence of Flavonoids was seen in chloroform and methanol extracts. The characteristic finding was that all the phytochemicals tested were present in methanol extracts expect Alkaloids, which was reported to be absent in all extracts.

 


Table.3: Qualitative phytochemical analysis of A.paniculata seed extract

 

Phytoconstituents

Sequential extracts

 

Methanol

Aqueous

Chloroform

Ethyl acetate

Hexane

Flavonoids

Alkaloids

Saponins

Tannins

Phenolic Compunds

Terpenoids

Glycosides

+

-

+

+

+

+

+

+

-

+

+

+

+

-

+

-

-

+

+

+

-

-

-

-

+

+

+

+

-

-

-

-

-

-

-

 - = absence and+ = presence of phytochemicals.


Table.3 shows the qualitative analysis of phytochemicals content of A.paniculata seeds. The result revealed the presence of most of the phytochemical compounds in methanol seed extract viz., Flavonoids, Saponins, Tannins, Phenolic compounds, Terpenoids and Glycosides. Aqueous extract reported the presence of all phytochemicals expect glycosides. Chloroform and ethyl acetate extracts reported the presence of tannins, Phenols and terpenoids. Flavonoids were present in aqueous, methanol and chloroform extracts. The hexane extract did not show positive for any of the tests. Alkaloids was found to be absent in all the solvent extracts. Yanishlieva16 reported on various pharmacological activities of flavonoids including vasodilatory, anti-carcinogenic, antiinflammatory, antibacterial, immune-stimulating, anti-allergic, antiviral and radioprotective effects.

 

In the present study, Phenolic compounds were present in chloroform, ethyl acetate, aqueous and methanol seed extracts of A. paniculata. They are found to be secondary metabolites with good antimicrobial properties as reported by Edreva et al.,17 Ali Ghasemzadeh et al.,18 reviewed that Phenolic compounds and flavonoids function as natural reducing agents, free radicals scavengers and quenchers of singlet oxygen formation. Further, findings of Dai and Mumper19 recorded that phenolics and flavanoids acts as good antioxidants and was found to be more effective than Vitamin C, E and carotenoids. These findings suggest that A.paniculata seed extract would act as an effective antioxidant agent, that contains the important phytochemicals viz., Phenols and Flavonoids.

 

Tannins are natural polyphenols, that have a characteristic strange odour and stringent taste and have been implicated to possess various pharmaco-therapeutic effect. Tannins derivatives are used as antimicrobials, antivirals, anti helmintics, antioxidants, and in cancer chemotherapy20. The above findings implicates that the seed extract could be used as an effective anti-inflammatory and skin ointment against allergies and infections.

 

Terpenoids were reported to possess various pharmacological activities such as anticarcinogenic, antiulcer, hepaticidal, antimicrobial activities21. Many natural derivatives of glycosides exhibits anti-inflammatory activity, inhibits gastric secretion and possess antimicrobial activity. The qualitative phytochemical analysis of A.paniculata revealed the presence of biologically important phytochemicals. With all the previous reported research findings, the current results of phytochemical composition suggest that A.paniculata seed extract could be used as a good remedial source for many diseases arising out to oxidative stress and other microbial infections.

 

Comparison of Tannin content of A. paniculata seed extract in different solvents:

The comparison of tannin content of seed extract of A.paniculata revealed (Fig.2) that the tannin content is recorded the highest in methanol extract (0.19±0.01), followed by aqueous extract(0.12±0.02) and chloroform extracts (0.11±0.02). The least value was recorded in the ethyl acetate extract (0.06±0.01).

 

Fig. 2. shows the tannin content of A. paniculata seed extract in different solvent extraction. The graph shows that the tannin content is recorded high in methanol, followed by aqueous and chloroform. The least value was recorded by ethyl acetate extract.

 

MIC of methanol extract of A. paniculata against different bacteria:

The observations of MIC study was recorded in Table.4. It was found that the minimum inhibitory concentration of the methanol extract was found to be varying between 10 -200µg/ml, with respect to most of the bacteria. The MIC of methanol extract for bacterial strains Salmonella typhi59, Vibrio cholera and Shigellaboydii 8 was found to be 100 µg/ml and for Bacillus licheniformis and Staphylococcus aureus, MIC was found to be 10µg/ml.

 

From the results of MIC studies, it is evident that the methanol extract of A.paniculata was found to possess antibacterial activity against gram positive and gram negative bacteria.


 

 

Table 4MIC of methanol extract of A.paniculata against different bacteria

Name of the Bacteria

Growth in broth containing different concentrations of A.paniculata seed extract (µg/ml)

0

10

25

50

100

200

Salmonella typhi59

Bacillus licheniformis 10341

Vibrio cholera 811

Shigellaboydii 8

Staphylococcus aureus ML-59

+

+

+

+

+

+

-

+

+

-

+

-

+

+

-

+

-

+

+

-

-

-

-

-

-

-

-

-

-

-

 All determinations were done in triplicates                       .

‘0’ – Control (without extract); ‘+’ – Growth; ‘-’ - No growth.

 


CONCLUSION:

Nature serves as the source of rich phytochemical diversity that possess important biological and pharmacological activities. In the current era of wide spread of various infectious diseases, there is a need to find out novel agents with therapeutic properties to rule out the adverse side effects of allopathic medicine system. The current investigation results has shown that A.paniculata seeds have rich phytochemical profile with good antibacterial activity. The phytochemical profiling provides a promising area of research in natural therapeutics and further studies are to be carried out to find the components responsible for its various pharmacological activities. The study will take a long way in analysing potent clinical and phytochemical applications of A.paniculata seeds.

 

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Received on 11.01.2019          Modified on 18.02.2019

Accepted on 20.03.2019        © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(5):2083-2088.

DOI: 10.5958/0974-360X.2019.00345.7