INTRODUCTION:

Herbal medicine significantly influences alternative therapeutic approaches, with many practitioners utilizing herbal remedies, Unani, and Ayurveda to treat patients. Remarkably, approximately 25% of all prescription drugs have roots in trees, shrubs, or herbs. India, often referred to as the medicinal garden of the world, boasts an abundant wealth of medicinal plants. One of the notable plants with historical and continued significance is Saraca asoca. Asoka, also known as Ashoka, derives from the Sanskrit word meaning "without sorrow" or "that which gives no grief." Universally recognized by its binomial Latin name Saraca asoca (Roxb.), De. wild, or Saraca indica, belonging to the family Caesalpinaceae, it is an evergreen tree known as the Asok tree in English. Referred to by various names across different languages and regions, such as Kankeli (Sanskrit), Ashoka (Assamese, Bengali, Gujrati, Hindi, Oriya, Punjabi), Ashokadamara (Kannada), Ashok (Kashmiri, Marathi), Asogam (Tamil), and Ashokapatta (Telugu), this plant is found throughout India, particularly in the Himalayas, Kerala, Bengal, and the southern region. Ashoka holds a special place in Hinduism, considered one of the sacred plants and especially revered by the Hindu God of Love, Kamadeva. A symbol of cultural and religious significance, it is worshipped annually on December 27th. Hindu mythology even mentions the Ashoka tree as the birthplace of Gautama Siddhartha (c.563-483 B.C.), the Indian philosopher and founder of Buddhism. The objective of this study is to provide comprehensive information about the medicinal and pharmacological importance of Saraca asoca, shedding light on its historical, cultural, and therapeutic significance.¹

Plants have served medicinal purposes since ancient times, with the traditional Ayurvedic system of medicine continuing to be widely embraced. Various factors, including population growth, limited drug availability, high treatment costs, adverse effects of synthetic drugs, and the emergence of resistance to existing treatments for infectious diseases, have heightened the focus on utilizing plant materials as a medicinal resource for a diverse range of human ailments. Among these plants, Ashoka stands out as one of India's oldest and sacred trees. Scientifically classified as Saraca asoca (Roxb.) De. Wilde and Saraca indica Linn., it belongs to the Caesalpinaceae subfamily of legumes.²

Scientific Classification:

Kingdom: Plantae

Division : Magnoliophyte

Class : Magnoliopsida

Order : Fables

Family : Caesalpiniaceae

Genus : Saraca

Species : asoca²

Bark of S. asoca:

The bark of S. asoca holds paramount medicinal significance, being recognized as the primary organ with therapeutic properties. This part of the plant is known to encompass a diverse array of compounds, including flavonoids, tannins, steroids, volatile oil, glycosides, and steroidal glycosides like β-sitosterol glucoside. Additionally, it contains various elements such as potassium, sodium, calcium, aluminum, strontium, iron, magnesium, and phosphate. The powdered bark also contains cellular components like tracheids, stone cells, parenchyma, sieve tubes, and other cell types. Moreover, lignin glycosides such as lyoniside, nudiposide, 5-methoxy-9-β-xylopyranosyl, isolariciresinol, and schizandriside, along with flavonoids like catechin, epicatechin, epiafzelechin-(4β-8)-epicatechin, procyanidin B2, deoxy procyanidin B, leucocyanidins, leucopelargonidin, and leucopelargonidin glucoside, have been identified in the bark of S. asoca. Furthermore, antioxidants like polyphenolics, gallic acid, and ellagic acid have also been reported from S. asoca bark.²

PHARMACOGNOSTICAL CHARACTERISTICS:

Microscopical characters:

The bark's transverse section reveals a periderm comprising a broad layer of cork, a radially flattened narrow cork cambium, and a wide secondary cortex with one or two continuous layers of stone cells interspersed with numerous patches of sclereids. The parenchymatous tissue within contains yellow masses and prismatic crystals. The secondary phloem is comprised of phloem parenchyma, sieve tubes accompanied by companion cells, and phloem fibers arranged in groups, with the presence of crystal fibers.²

Description:

A. Color Brown

B. Odor Characteristic

C. Taste Characteristic

D. Appearance Free flowing powder¹

Phytochemistry:

Saraca asoca (Roxb) Willde, a member of the Fabaceae family, is commonly known as the Asoka tree. A comprehensive investigation was undertaken to identify the bioactive compounds present in the bark of Saraca asoca using Perkin-Elmer Gas Chromatography – Mass Spectrometry. The mass spectra of the compounds detected in the extract were cross-referenced with the National Institute of Standards and Technology (NIST). The GC-MS analysis of the ethanolic extract from Saraca asoca bark revealed the presence of sixteen compounds. Among these, the predominant compounds include 1,3-Benzenediol, 4-propyl- (30.03%), 2-Hydroxy-5-methylisophthalaldehyde (22.06%), Homovanillic acid (8.60%), Methyl 4-O methyl-d-arabiopyranoside (7.34%), Methoxy olivetol (6.79%), n-Hexadecenoic acid (6.00%), while minor compounds encompass β-Sitosterol (4.04%), 9,12-Octadecenoic acid (Z, Z)- (3.41%), 9-Octadecenoic acid (Z)-, methyl ester (2.45%), Isoquinolin-6,7-diol-1-carboxylic acid, N-acetyl-l-methyl- (0.77%), and Butorphanol (0.62%). These findings present Saraca asoca bark as a potential herbal alternative for various diseases.³

Helminthiasis stands as the prevalent infection caused by worms, contaminating various human body parts. Typically, these worms inhabit the gastrointestinal tract, liver, and other organs. Presently, anthelmintic drugs like albendazole, mebendazole, thiabendazole, niridazole, diethylcarbamazine, ivermectin, and praziquantel are extensively employed to manage helminthiasis. However, these drugs come with significant drawbacks, including hepatotoxicity, loss of appetite, dizziness, nausea, vomiting, abdominal pain, headache, and diarrhea. Hence, the quest for more effective anthelmintic drugs with minimal side effects is imperative. A substantial 80% of the global population relies on traditional medicines and plant extracts, utilizing their active constituents to address primary healthcare needs.⁴

MATERIAL AND METHOD:

Plant material - Collection and Processing:

The stem bark of Saraca asoca was gathered from Srirangam, Tiruchirappalli, Tamil Nādu, India, and its authenticity was verified by Dr. John Britto, a professor in the Department of Botany at St. Joseph College, Tiruchirappalli. A voucher specimen was duly deposited in the Department of Microbiology, Srimad Andavan Arts and Science College, Tiruchirappalli, India. The stem bark underwent shade drying and was subsequently powdered using a mechanical blender from Smith, India. The resulting dried powder was stored in an airtight container and later utilized for extraction purposes.⁵

Processing of plant material:

Preparation of crude powder:

The gathered plant material underwent manual cleaning to eliminate coarse impurities, following which it was air-dried in shade at a well-ventilated section within the laboratory. The dried bark was subsequently crushed and ground using an electric mixer-grinder to produce crude powder, which was then stored in airtight containers or poly bags (Azwanida, 2015; Odey et al).⁶

Extraction:

Extraction is a frequently utilized method for extracting active substances from a crude drug, utilizing various solvents. Successive solvent extraction is a method where the drug is sequentially extracted with solvents of varying polarity, transitioning from a non-polar solvent to a more polar one. The diverse extractives obtained from the crude drug serve as approximations of its chemical constituents.⁷

1. Cold maceration: A quantity of 50g of crude powder from each plant material was immersed in 400ml of analytical grade methanol within a glass flask, covered with aluminum foil. The mixture was stirred at hourly intervals at room temperature, allowing it to soak for a duration of 72hours. After soaking, the crude powder was filtered through Whatman filter paper No.1 using separating funnels. The resulting filtrates were concentrated through evaporation.⁶

2. Solvent extraction: A quantity of 24grams of powdered plant material underwent pressurized sequential extraction using an accelerated solvent extractor (ASE 150, Dionex Inc., USA). The powder was mixed with diatomaceous earth at a ratio of 4:1 and placed in a 100ml sample cell, which was then loaded onto the system. Extraction took place under 1500 psi pressure at 60°C, with a flush volume of 90%, employing three static cycles and HPLC grade ethanol. This process was repeated multiple times to obtain the required extract amount¹². Subsequently, the solvents were evaporated using a rotary evaporator (Buchi, Switzerland). A portion of the dried extracts was weighed for the isolation of the marker compound, while the other portion was dissolved in DMSO to achieve desired concentrations for cell line studies.⁸

Phytochemical analysis of extracts:

The residue extracted from each plant was subjected to testing to determine the presence of various phytoconstituents, including alkaloids, tannins, flavonoids, saponins, glycosides, resins, triterpenes, reducing sugars, and proteins. Standard procedures were employed for these tests.

Test for alkaloids:

0.5 to 0.6g of various extracts were mixed in 8ml of 1% HCl, warmed and filtered. 2ml of the filtrate were treated separately with both reagents (Wagner’s and Dragendorff).

a) Dragendorff reagent: The filtrate of the extract was added to the reagent and development of turbidity of precipitation was considered as the presence of alkaloid.

b) Wagner’s reagent: The filtrate of the extract was added to the reagent and development of brown flocculent precipitate indicated the presence of alkaloids.

Test for glycosides:

The solution obtained in Benedict’s test was filtered and diluted HCl was added. Equal quantity of Benedict’s reagent was added and boiled. Appearance of brownish precipitate revealed the presence of glycosides.

Test for tannins:

Methanol was added to the residue of the extract. The solution was heated and filtered through Whatman filter paper. Filtrate obtained was treated with different reagents.

a) Lead acetate test: 2-3 drops of lead acetate solution was added to the above-mentioned extract solution. The formation of precipitate indicated the presence of tannin.

b) Ferric chloride test: Few drops of ferric chloride solution were added to the above filtrate. A green colouration in the filtrate of the methanolic extract indicated the presence of tannin.⁶

Thin layer chromatography (TLC) TLC was run for the crude sample and the mobile phase solvent was standardized using different ratios of solvents. Ethanolic extract of the sample was run with 100% chloroform. Petroleum ether extract of the sample was standardized for column chromatography and separated using TLC were 6:4 (petroleum ether: ethyl acetate) was standardized as the mobile phase⁹

Column chromatography (CC) Column chromatography for ethanolic extract The column was prepared as mentioned above with 100% chloroform as mobile phase. Filter dried ethanolic extract of the sample was mixed with the solvent and silica gel. It was dried using a water bath and the dried extract with silica was scrapped and put into a butter paper and it was loaded onto the top of the packed column. Once after the sample was loaded, the solvent was poured and the set up was observed for separation of fractions. The fractions were collection in test tubes and TLC for each fragment were performed to check the purity of the fractions and the Rf values were calculated by the formula, The solvent was allowed to evaporate and the dry weight of the fractions was noted.⁹

Taxological Acute and sub-acute oral toxicity study: Toxicity study was performed according to the Organization of Economic Co-operation and Development (OECD) guideline for testing of chemicals (OECD 2001). In order to study acute toxic effect of the plant extract 7 groups of 10 animals (5 male and 5 female) were used. Group I used as control and II to VII used as treatment groups. Last six groups were received a single oral dose of 1, 2, 3, 4, 5 and 6 g/Kg body weight of the methanolic extract respectively for acute toxicity study. All the mice were fasted overnight prior to treatment and body weight of each of them were recorded. All the animals of the last six groups were administered the respective amount of methanolic extract dissolved in 1 ml of distilled water, the control group of animals was administered only distilled water through gastric tubing. On the 15th day, all mice were kept fasted for 15 h, and then sacrificed for necropsy examination. The internal organs were excised and weighed. The gross pathological changes of the tissues were studied and LD50 value was determined. The selection of doses for mice used to study sub-acute toxicity was determined from the equivalence of 15-120 times of normal human dose (10-20 mg/Kg) that also in accordance to the information collected from herbalists. The low dose was 0.3g/kg and the higher dose was selected as 1.2g/kg. The extract was administered daily for 30 days to the mice of both low dose group and higher dose group (each containing 5 male and 5 female animals) through gastric tubing and the control group containing the vehicle. On the 31st day mice were made fasted for 15h and sacrificed. Blood samples for hematological blood analyses were taken by puncturing heart. The internal organs were weighed to determine relative organs weights, and examined for gross lesions. All tissues were fixed in Buins solution for histological examination.^10.

RESULT AND DISCUSSION:

Anthelmintic:

Saraca indica extract has been used for anthelmintic activity, the extractions were prepared to obtain 1, 2.5 and 5% concentration of the standard anthelmintic drug like Piperazine citrate. Experiments showed that the ethanolic extracts were relatively more potent as an anthelmintic agent due to presence of alkaloids. The methanolic extracts are effective probably due to the involvement of glycosides, tannin, flavonoids and terpenoids seems to be the accountable phytochemical constituent for signifying anthelmintic activities of extracts.¹¹

Table 1: Extract ability and physical properties of Saraca asoca bark and Azadirachta indica seeds⁶

S. No	Physical properties	*S.asoca* bark
S. No	Physical properties	Methanolic extract	Aqueous extract
1	Color	Brownish black	Dark brown
2	Consistency	Solid	Solid
3	Extractability	4.29%	3.72%

Table 2: Phyto chemical analysis of Saraca asoca bark extracts⁶

S. No	Active Principle	Test Applied	Result
S. No	Active Principle	Test Applied	Methanolic	Aqueous
1	Alkaloids	a) Dragendorff test b) Wagner’s test	Negative Positive	Negative Negative
2	Reducing sugars	Benedict’s test	Positive	Positive
3	Glycosides	Benedict’s test	Negative	Negative
4	Tannins	a) Lead acetate test b) Ferric chloride test	Positive Negative	Positive Positive
5	Resins	Hydro alcoholic extract solution in distilled water	Negative	Negative
6	Saponins	Foam test	Positive	Positive
7	Sterols	Salkowski test	Negative	Negative
8	Fixed oils	Filter paper test	Positive	Positive
9	Proteins	Biuret test	Negative	Negative
10	Anthroquin ones	Bontrager’s test	Negative	Negative
11	Flavonoids	Addition of diluted NaOH solution and diluted HCl	Negative	Negative

Table 3: Determination of LD50 values by Miller and Tainter method ²² of the methanolic extract of Saraca asoca bark in mice.

Oral dose (mg/kg)	Log dose	Dead/total	%Death	Prob it
1000	0	0/10	0	0
2000	3.3	0/10	0	0.078
3000	3.47	1/10	10	0.796
4000	3.6	3/10	30	2.448
5000	3.69	3/10	30	4.463
6000	3.77	7/10	70	6.252

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Received on 02.05.2024 Modified on 12.06.2024

Research J. Science and Tech. 2024; 16(3):171-175.

DOI: 10.52711/2349-2988.2024.00025