Anti -Asthmatic Activity of An Ethanolic Extract of Entire Plant of Euphorbia Hirta from the Pharmacognostical, Pharmacological and Phytochemical Screening - An Research

Euphorbia hirta, commonly known as the “asthma plant,” is a medicinal herb renowned in traditional medicine for its diverse health benefits, particularly in managing respiratory ailments like asthma. This is also known as the Australian asthma herb or Queensland asthma weed, spurge or milkweed.  This small, green, bushy plant and it is a common weed found throughout the hotter parts of India and most of the tropical and subtropical countries It is frequently seen occupying open waste spaces, grasslands, roadsides, pathways and is often dismissed as a weed. Euphorbia plants are characterized by the presence of milky latex, which they exude when broken.  The extract of Euphorbia hirta has a sedative effect on the mucous membrane of the respiratory and genitourinary tracts. The whole plant also has been reported to possess anti-asthmatic, antiinflammatory, anti-bacterial, anti-amoebic, antifungal, antiviral, spasmolytic, anti-diarrheal, anxiolytic, analgesic, anti-pyretic, anti-malarial, and anti-hypertensive properties. Euphorbia hirta Linn has been long utilized as an herbal medicine globally in remedying and curing many infections and diseases in humans. Euphorbia hirta is especially notable for its role in treating respiratory conditions such as asthma, bronchitis and other lung-related ailments. A putative species has been known for its medicinal, cultural and ethnobotanical uses.  In India, the plant parts are traditionally used to treat worm infestations in babies and for dysentery, gonorrhea, jaundice, acne, pimples, digestive disorders, cancer, diabetes and different tumors. The extracts of the root are used against vomiting and diarrhea and for snakebite as an anti-venom. It is not toxic when taken in typical dosages. The effectiveness of E. hirta in treating asthma may lie predominantly in the synergistic relationships between the flavonoids, sterols and triterpenoids.  The medicinal usefulness of this herb has been the subject of numerous chemical and microbiological studies. According to the WHO estimates, Since 1970s, the global prevalence, morbidity, mortality and economic burden of asthma have increased particular in children released in December 2016, there were 3,83,000 deaths due to asthma in 2015.   Asthma is an obstructive pulmonary disease with different etiologies, affecting approximately 300 million people worldwide. By 2025, a further 100 million will be added to this estimate. The plants medicinal use dates back centuries, with historical records from different continents documenting its therapeutic roles. (FIG. 1)

Fig.1 The Figure Shows the Whole Plant of Euphorbia Hirta

1.1. Plant Profile

Synonym

Asthma Weed, Snake Weed, Hairy Spurge, Ara Tanath

Biological Source

It consists of fresh and dried whole plant of Euphorbia hirta Linn

Family   Euphorbiaceae

Geographical Source

India  

• Africa
• Australia
• Asia

Taxonomical Classification

Botanical Source

5. Leaves
5. Flowers
5. Fruits And Seeds
5. Stem
5. Root

Leaves

• The leaves are simple with a very short petiole and are arranged oppositely distichous. These may be green or red in color and sometimes with a purple blotch on the adaxial side. Both sides of the leaf are covered with sparsely appressed pubescent. A white latex flow when cut.(FIG.2)

• The blade is lanceolate-oblong, long elliptic, or ovate-lanceolate in shape. Its base is asymmetric, having obliquely rounded on one side and cuneate on the other side. In addition, the apex is acute and the margin is serrate to serrulate. The venation is trinerved at the base. It has 3–5 pairs of lateral veins arising at the base or slightly above the base initiated along the blade.

Fig. 2 The Figure Shows the Leaves of Euphorbia Hirta

Flowers

• The inflorescence called ‘cyathium’ is present. It is clustered in dense cymose situated at the terminal or at the upper nodal regions of the stem.
• The flowers are monoecious; androecium and gynoecium are assembled in the same companulate involucre. Each involucre contains one female flower surrounded by a few male flowers.
• Frequently, the pedicelled cyathia are green and often tinged reddish; all parts are very hairy, consisting of 20-30 clusters in the axil. Also, 4 glandular structures with a petallike appendage are present in the involucre. (FIG.3)
• It appears ovate on the transverse top view. 4-5 staminate florets are present in the cyathium. These florets are sessile with pedicles devoid of hair (glabrous). Also, a bristlelike bracteole is present, and the anthers are sub globose. Similarly, the perianth is absent in the florets.
• Moreover, a pistillate flower is exserted in the involucre. Like the male flower, the perianth is also absent. The ovary is superior, consisting of 3 angular locules that are sparsely pilose. The styles are free from the base, deeply bifid, and the stigma is capitate.

Fig. 3 The Figure Shows the Flowers of Euphorbia Hirta

Fruits And Seeds

• Euphorbia hirta has allomorphic pistillate fruits.
• The fruit is exerted, 3-lobed, base truncate, covered with short hairs. (FIG.4)
• The seeds are oblong, 4-sided, prismatic, wrinkled, and brownish pink, with a 3-seeded capsule; it is green and covered with fleshy prickles, with smooth, hard, mottled, crustaceous testa with a white caruncle at the top enclosing oily endosperm.

Fig. 4 The Figure Shows the Fruits and Seeds of Euphorbia hirta

• The stem of E. hirta is caulescent. It is erected, ascending to decumbent.
• It is cylindrical and often reddish or green in appearance and growing to 10-75 cm long.
• The stem has a strong pubescent adpressed vestiture; yellow-brown uniseriate multicellular trichomes are growing 1.5 mm shorter or longer. The base is woody and sparingly branched in a monopodial pattern at the middle.
• The internode can grow with an average length of 2.49 cm. The nodes are a bit thickened, characterized by the presence of small membranous linear stipules that shed off at an early stage. Milky latex is flowing when the stem is cut. (FIG.5)

Fig. 5 The Figure Shows the Stem of Euphorbia hirta

Root

• The root of E.hirta is a typical taproot system, growing positively geotropic.
• The primary root is dominant, distinct, and enlarged at the base of the stem.
• Generally, the primary root is cylindrical in shape, by which lateral roots (the secondary and tertiary roots) are arising and spreading below the ground and laterally. These root characteristics are typical of eudicots. (FIG.6)

Fig. 6 The Figure Shows the Root of Euphorbia hirta

Medicinal Uses

• Euphorbia hirta has the potential to cure various respiratory disorders like asthma and bronchitis.
• Latex of plant helps to heal warts fastly.
• In South India, it is used as ear drops in the treatment of boils, sores, and wounds.
• The whole plant is decocted for athlete’s foot,enteritis, fever, gas, itch, and skin conditions.
• The extract of the plant used as an antidote for snakebites and scorpion stings.
• It is used for relieving toothache, severe headache, rheumatism and colic.
• When decoction of the herb is consumed, mothers get benefited by increased production of milk and has been used traditionally to address various women’s health issues, including menstrual disorders and postpartum recovery.
• Skin infections like rashes, warts and swelling take the milky extract of the herb and apply it over the affected area. The skin gets cured very soon.
• For a sore eye, apply the latex to the eyelids below the eyes
• Euphorbia hirta is commonly used to treat diarrhoea and dysentery. (FIG.7)

Fig. 7 The Figure Shows the Medicinal Uses of Euphorbia hirta

The National Institute of Health defines asthma as a chronic inflammatory disorder of airways in which cellular elements play a major role particularly mast cells, T-lymphocytes, eosinophils, epithelial cells and neutrophils.  Asthma is a life-threatening, enduring respiratory disorder, resulting in the inflammation and narrowing of airways. Exacerbation includes bronchospasm, edema, mucus secretion, cellular infiltration and damage of the airway epithelium. Asthma is associated with wheezing, breathlessness, chest tightness, and coughing brought about by inflammation, bronchial constriction, and excessive mucus secretion due to bronchial hyper-responsiveness. 10 % of asthma sufferers are children and 5% are adults. (FIG.8). Asthma mortality in the United States amounted to some 5000 persons in the year 2000. The global prevalence of the disease is expected to rise to 400 million by the year 2025. Most of the available pharmacological therapies simply control the known mechanisms by which the disease occurs; there is no complete cure for asthma. Pharmaceutical products have been the main source of Anti-asthma treatment, but some of these have recently been associated with serious adverse effects. The increased number of asthma patients in every country led to drug unavailability, expensive drug prices and exorbitant. In India, Ayurveda is a traditional practice of medicinal plants in the treatment of asthma that is considered to be natural and safe (TABLE.1 & 2)

Fig. 8 The Figure Shows the Human Normal and Asthmatic Lung

Mechanism Of Action 

Asthma is well known to have a high level of IgE that binds to the receptors of mast cells and inflammatory products. The interaction between antigen and antibody IgE results in the activation of an inflammatory cellular reaction.   Thereby releasing the mediators, such as histamine and prostaglandins, that ultimately lead to the contraction of airway smooth muscles. Asthma can be triggered by various factors such as viral respiratory infections, certain chemicals, certain medications, airborne allergens, occupational sensitizers, smoke, air pollutants, stress, anxiety or extreme emotional arousal, may also trigger asthmatic attacks. (FIG.9)

Fig. 9 The Figure Shows the Mechanism of Action for Asthma

1. Extrinsic asthma

2. Intrinsic asthma

1.Extrinsic asthma

It is caused by allergic responses to such things as house dust, animal fur, or various foods. Such cases account for about 10 to 20%.

2.Intrinsic asthma

It is caused by genetic and structural problems and infection. Both physiological and psychological. Such cases account for about 50–60%.

Causes

Asthma can develop when the body’s defence system reacts strongly to a new substance in the lungs. Asthma usually develops during childhood (called childhood asthma) and some people do not show signs of asthma until adulthood (adulthood asthma). Many factors like allergens in the environment, viral infections, and family history work together to develop asthma. 

• Family history: Asthma is more likely to develop when there is a family history of asthma that is if one of the family members, like a father or a sibling, has asthma.
• Allergy conditions: Patients with allergic conditions such as eczema and rhinitis are more prone to develop asthma. 
• Lifestyle factors: A sedentary lifestyle in urban areas increases the prevalence of asthma.
• Development of the lungs can be affected by certain events in childhood such as low birth weight, prematurity, exposure to smoke, and air pollution. When the lungs are not developed fully, they do not function effectively and increase the prevalence of asthma.
• Being overweight or obese in children or adults also causes asthma.
• Exposure to environmental allergens and irritants including outdoor and indoor, air pollution, dust, mites, molds, and exposure to chemicals in occupational areas also increases the risk for the development of asthma

Symptoms

Symptoms of asthma occur intermittently and last for about a few hours to days. These symptoms resolve when the triggers are removed or with the administration of medications. 

Common asthma signs and symptoms 

• Shortness of breath (SOB)
• Cough
• Tightness of chest
• Decrease in peak flow meter 
• Waking up at night due to a worsening of cough

Severe asthma symptoms

Symptoms of severe asthma are often persistent, and these symptoms are observed at least once a day.     

• Breathlessness which is persistent and continuous
• Heavy feeling in the chest
• Wheezing
• Severe cough
• Difficulty in speaking
• Allergic responses to pollen and dust
• Extreme sensitivity to scents and smell
• Blue lips or fingers
• Rapid heart rate 
• Drowsiness
• Confusion ? Dizziness

Medical Condition 

Those who are suffering from eczema or hay fever is on high risk of developing asthma.

Diagnosis

• Physical examination: Where your doctor checks breathing with the stethoscope.
• Breathing test: A pulmonary function test measures airflow into and out of your lungs.
• Spirometry: Measures how much air you breathe out and how fast. Some people feel light-headed or tired from the breathing effort for this test.
• Peak expiratory flow (PEF) tests: Measure how fast you can blow air out using maximum effort.

The test can be done during spirometry or with a small handheld device.

• Bronchodilator responsiveness tests: Measure how much better you can blow air after inhaling a fast-acting bronchodilator, a medicine that helps open your airways by relaxing the muscles around them. The test compares your spirometry or PEF results before and after the medicine
• Allergy tests: It can find which allergens, such as pet dander or pollen, cause a reaction in your body when you are around them. Your provider may run these tests if you have a history of allergies.
• Blood test: It can measure levels of white blood cells, which can indicate inflammation. Blood tests can also find your allergies.
• Chest CT scan: It may show whether the walls of your airways are thicker.

2. MATERIALS AND METHODS

• Source of the Plant Material
• Collection of the Plant Material
• Extraction

? Pharmacognostical Studies

• Macroscopical studies
• Microscopical studies 

• Phytochemical Screening
• Analytical Methods ? GC/MS

? Invitro Studies

MTT Assay Using THP – 1 Cell Line

2.1 Source of The Plant Material

Synonym 

Asthma Weed, Snake Weed. Hairy Spurge, Ara Tanath, 

 Family

Euphorbiaceae

Vernacular Names 

• Arabic: Labeinah, Em elhaleeb, Euphorbia
• Bengali: Barokhervi 
• English: Pill-bearing spurge, asthma weed, asthma herb, snakeweed 
• Hindi: Dudhi 
• Malayalam: Nelapalai 
• Marathi: Dudnali, govardhan
• Philippines: Botobotonis
• Sanskrit: Amampatchairaisi, barokheruie, dugadhika
• Tamil: Amumpatchaiyarissi

Geographical Source

• India – Bihar, Gujarat, West Bengal Uttar Pradesh, Madhya Pradesh, Himachal Pradesh
• Africa
• Australia
• Asia

2.2 Collection of the Plant Material

The Plant Euphorbia hirta Linn was collected from Lalgudi in Tiruchirapalli District, and Authenticated by Prof. Dr. V. Nandhagopalan, Head of the Department of Botany, National College, Trichy.

2.3 Extraction

Preparation Of Plant Powder

• The plant material was shade dried for about four weeks. 
• Then the dried material was crushed to coarse powder using an electrical grinder and stored in airtight bottles. The powder was used for further analysis.

Preparation Of the Ethanolic Extract

• Dried powder of plant material was placed in thimble chamber of the Soxhlet apparatus.
• The extraction solvent ethanol was heated in the bottom flask, vaporizes into the sample thimble, and condenses in the condenser and drip back. 
• About 100 g of dried sample powder was weighed and extraction process is carried out by using 250 ml of ethanol in Soxhlet apparatus for 48 hr. 
• When the liquid content reached the siphon arm, the liquid contents emptied into the bottom flask again and the process was continued. 
• The extract was concentrated by evaporation at 70 °C for 8 hrs and then dried. (FIG.10)

Fig.10 The Figure Shows the Identification of Extract of Crude Drug

 Macroscopic Studies 

The Macroscopy was carried out to determine the Nature of the plant, Organoleptic Properties (Colour, Odour, Taste, Size, Shape and Extra features) of the Whole Plant.

Leaves

• Colour: Dark green above, pale beneath.
• Odour: Odourless 
• Taste: Bitter
• Size: 1 - 2.5 cm long
• Shape: Ovate

Flowers

• Colour: White/pinkish petal-like appendages 
• Odour: Odourless
• Taste: Bitter

Fruits

• Colour: Yellow
• Odour: Odourless
• Taste: Bitter
• Size: 1 - 2 mm in diameter

Seed

• Colour: Pinkish brown
• Odour: Odourless
• Taste: Bitter
• Size: 0.57- 0.70mm long, 0.065mg/seeds
• Shape: Oblong

Stem

• Colour: Reddish or Purplish, green
• Odour: Odourless
• Taste: Bitter
• Size: 10-75 cm long
• Shape: Cylindrical

Root

• Colour: Light Brown
• Odour: Odourless
• Taste: Bitter

Extra Features

Leaves - Elliptic-oblong to oblong-lanceolate, acute or subacute 

Flowers - Male flowers-sessile, with linear bracteoles, solitary stamen, are fringed and lack a    perianth.  Female flowers-a short pedicel, superior ovary, ringed perianth, three styles threecelled, minute, and two-    fid apex hair

Fruits - three-celled, hairy, keeled capsules, containing three brown, four-sided, angular,                                    wrinkled seeds

Seeds - Four-sided, without caruncle, slightly wrinkled.

Stem - Smooth, slender, densely branched, hairy stems & posseses 2-3 internodes.

Root - Arranged in a taproot system

Microscopic Studies

Transverse Section of Leaf (Fig.11)

Nodal anatomy 

One median and two lateral leaf traces are observed in the nodal region with three leaf gaps for a leaf. Node is trilacunar three trace.

Lamina 

• Lamina is dorsiventral. Single layer of palisade cells is present on adaxial side and spongy cells are arranged in 4 - 5 abaxial layers. 
• Lamina has smaller collateral strands in the middle region, which are surrounded by latex cells. Latter contain chloroplasts and appear green. 
• Lamina is amphistomatic with anomocytic stomata on both surfaces. The stomatal apertures are field up with cutin in many stomata. 
• Wall of epidermal cells is highly sinous on both surfaces.

Midrib 

Midrib has parenchymatous ground tissue except adaxial region where palisade cells of lamina are extended.   A single large roundish collateral strand is present in the center, which is completely or partially encircled by latex cells.

Petiole

Petiole has parenchymatous ground tissue and three small collateral vascular bundles arranged in a crescentic manner. Scattered latex cells are present in the ground tissue of petiole. Trichomes similar to the stem are present in lamina, midrib and petiole.

Fig.11.  The Figure Shows Transverse Section of Leaf

Transverse Section of Stem (FIG.12)

In young stem 16 - 18 collateral vascular bundles are observed in a ring. Pith is made up of parenchyma cells. Pericycle is formed by alternate groups of fibers and parenchyma cells.   Epidermis is consisted of small rectangular or tubular cells on which a thick and wavy cuticle is present. Hypodermis of collenchyma is observed below epidermis   Epidermal surface is covered by large number of Glandular, multicellular, uniseriate, filiform, cylindrical and bent trichomes.Stem has cortex of 6 - 8 layers. Scattered latex cells are present in the cortical region in old stem secondary vascular tissues are produced. Secondary xylem has solitary vessel elements or found arranged in radial groups of 2 - 4. Xylem fibers are abundant and xylem rays are uni- biseriate.

Fig.12 The Figure Shows Transverse Section of Stem (Lc-latex cell, Co-collenchyma, Ux- Uniseriate xylem rays)

It is composed of cortex of 4 - 6 layers of parenchyma and tetrarch xylem. Rarely xylem is diarch. Pith is scanty or absent. Vascular cambium forms larger amount of thick walled, polygonal or rectangular fibres, roundish, angular, solitary vessel elements and uniseriate rays. The secondary phloem appears deformed or compressed.

Fig.13 The Figure Shows Transverse Section of Root (xf- xylem fibres, ux- uniseriate xylem rays, pd- phelloderm, sp- secondary phloem)

Test For Alkaloids 

Wagner’s test:

2ml of plant extract was taking, 3-5 drops Wagner’s reagent was added, and the formation of Reddish-brown precipitate or coloured was observed.

Test For Saponins

Foam test:

Take 2 ml of extract and 5ml distilled water and shake vigorously. Observe for the formation of Persistent foam for 10-15 minutes that confirms the presence of saponins.

Test For Flavonoids

Alkaline reagent test: 

2 ml of plant extract was taken and treated it with 3-5 drops of 20% sodium hydroxide solution. Observe for formation of intense yellow colour, which becomes colourless on addition of 0.5ml dilute Hcl indicates presence of flavonoids

Test For Terpenoids 

Take 2 ml of plant extract, treat it with 0.5ml of conc. HCl, and indicate for the formation of yellow precipitate or coloration.

Test For Tannins

Braymer’s Test:

Take 2 ml of extract, treat it with 1ml of 10% Alcoholic Ferric Chloride solution, and observe for the formation greenish colour.

Test For Steriods

Add 2 mL of extract to 2 mL of chloroform and 2 mL of concentrated H2SO4. A reddish colour indicates steroids.

Test For Amino Acids

Million’s Test:

Take 2 ml of extract, add 5 drops of Million’s reagent, and warm the mixture in boiling water bath for 10 minutes. Cool to room temperature and add 5 drops of Sodium Nitrate solution. Observe the Brick red colour it indicates the presence of Amino acids.

Test For Volatile Oils

Take 2 ml of extracts, 0.5 ml of dilute NaOH and small amount of dilute HCl acid were added and the formation of white precipitates indicates volatile oils.

Test For Carbohydrates

 Take 2ml of extracts in a test tube, add 2 drops of 1% α 1- naphthol, mix and then allow 2ml of conc. Sulphuric acid to flow down the side of the inclined tube to layer under the mixture.

Test For Proteins

Biuret Test:

Take 2ml of the extract, egg albumin and deionised water in the test tube. Add 2ml of biuret reagent to the test tube. Shake well and allow the mixtures to stand for 5 minutes. Observe no colour change that is solution remains blue proteins are not present.

Fig.14 The Figure Shows the Qualitative Phytochemical Screening

Gas Chromatography/Mass Spectrometry (Gc/Ms) Analysis

The extract of plant was tested by chromatography–mass spectrometer analysis to detect and quantify the components using GC ultra-system from Thermo Fisher Scientific, Waltham, MA, USA, fitted with a 30 m × 0.25 mm × 0.25 m Elite-5-MS capillary column, and was used to analyze the extracts. The temperature of the column during the analysis was raised at a rate of 4  °C/min from 40 °C to 220 °C. The injection volume of 1 μL was maintained at the injector temperature of 250 °C; the transfer temperature was maintained at 280 °C and at the flow rate of 20 mL/min, with helium as the carrier gas. The following MS settings were used: EI mode, 70 eV for the ionization voltage, a range of scan of 50–600 Da, and 180 °C for the ion source temperature. Tentative identification of the peaks was achieved based on a library search using Wiley Registry 8 Edition and NIST.  (GRAPH.1)

Graph.1. Gas Chromatography/Mass Spectrometry (GC/MS) Analysis

Cell Culture and Differentiation of Thp-1 Cell Line (Starr Et Al. 2018)

THP-1 cells were obtained from NCCL, Pune. Cells were cultured in (RPMI-1640) medium accompanied with 10% complement-inactivated fatal bovine serum (FBS), 100 IU/mL penicillin, 100 μg/mL streptomycin, and 3.7 g sodium bicarbonate/L (Sigma-Aldrich,). In order to activate THP-1 (4x10⁴) cells to differentiate, cells were cultured in 96-multi-well culture plates, then 100 ng/mL of phorbol 12-myristate 13-acetate was added. Cells were washed with RPMI-1640 serumfree medium prior to each experiment to remove undifferentiated cells. For activation of macrophage-like cells, cells were then treated with LPS (100 ng/mL) for 24 h.

Analysis Of Cell Viability (MTT Assay)

Cell viability was measured by MTT assay. THP-1 cells were exposure to different concentrations (5 to 100 µg/mL) of crude extract. Briefly, media was replaced with 50 µL/well serum-free media and 50 µL/well MTT reagents. Cells were cultured in 24-well plates and incubated for three hours. Optical density (OD) was measured at 590 nm using a microplate reader (Spectra Max). All experiments were performed in triplicate.  (FIG.15).

Fig.15 The Figure Shows MTT Assay

Extraction Yield 

The Ethanolic extract of Euphorbia hirta yielded 2.76%.

Pharmacognostical Studies

Macroscopical Studies

The macroscopy was carried out to determine the nature of the plant. 

Organoleptic properties of the whole plant was identified.

Microscopical Studies

The Microscopical characters was presented the transverse section of stem, leaf, root 

• Latex cells: Present in the lamina, midrib, petiole, stem cortex, and root.
• Trichomes: Found on the stem, lamina, midrib, and petiole.
• Vascular bundles: Collateral type in leaf, stem, and petiole.
• Stomata: Anomocytic and amphistomatic in the leaf.
• Secondary growth: Observed in old stem and root.

Phytochemical Screening

The chemical constituents present are Alkaloids, Saponins, Flavonoids, Terpenoids, Tannins, Steroids, Amino acids, Volatile oils, Carbohydrates, Proteins (TABLE.3)

Table.3 Qualitative Phytochemical Screening

Gas Chromotography/Mass Spectrometry

GC/MS method is used qualitative identification quantitative measurement of individual components in a complex mixture. (TABLE.4)

In Vitro Anti-Asthmatic Activity (Table.5 & 6) (Graph.2) (Fig.16)

Table.6 Statistical Analysis of Cell Proliferation For 48h

Graph.2. Percentage Of Cell Viability

Fig 16 The Figure Shows Microscopic Image of Cell Proliferation