INTRODUCTION:

Both traditional as well as modern medicine make use of an increasing number of medicinal plants these days to treat a wide range of ailments. This is because herbal products are safer than synthetic ones, which are thought to be harmful to people and the environment. Several plant components, including the leaf, root, flower, along with seed, have physiologically and medicinally active secondary principles that put to use to make medications. S. laurina Dalzell, a member of the family Annonaceae; Laurel bow-wood in English; Sagare; Harikinjali in Kannada; as well as Sajeri in Marathi, are some of the common names. It is endemic to the Western Ghats of India. The vast array of indigenous plant species found in the Western Ghats makes it one of the world's mega-biodiversity hotspots. This plant is found in a canopy tree species of evergreen forest in the Western Ghats. In the Indian Western Ghats, the S. laurina Dalzell plant has been deemed to be a near threat or at lesser risk.¹

Leaves, bark, and seeds of S. laurifolia, a very endangered species, are used by tribal people as a remedy for peptic ulcers and diarrhea. Leaves have therapeutic properties. Tribal peoples consume ripe fruits of S. laurifolia, and leaves are used to alleviate rheumatism. Treatment for AIDS disease involves the use of the genus Sageraea.² Diverse molecules with biological activity, including carbohydrates, proteins, enzymes, quinones, terpenoids, flavonoids, sterols, simple phenolic glycosides, carotenoids, alkaloids, polyphenols, saponins, vitamins, minerals, lipids, and oils, exist in medicinal plants.

These highly significant bioactive compounds from plants, which are vital as medications, primarily demonstrate pharmacological actions.³ The natural defense mechanism against microbial, noxious chemical, or physical trauma-induced tissue damage is inflammation. It is a ubiquitous defense mechanism executed by the innate as well as adaptive immune systems to fend off pathogenic invaders. It is generally characterized as a nonspecific reaction to tissue dysfunction.⁴ Chemical mediators’ liberation from disabled tissue as well as migratory cells initiates body's response to allay or destroy invasive organisms, eliminate irritants, as well as prepare ground for tissue restoration.⁵ Redness, swelling, heat, discomfort, and impairment of function are its four cardinal warning signs.⁶

Acute and chronic inflammation are the two different forms that exist. Acute inflammation is a transient reaction that typically leads to healing: leukocytes infiltrate the injured area, eliminating stimuli and mending tissue. Contrarily, tissue death, active inflammation, and measures for tissue healing are all hallmarks of chronic inflammation, which is a prolonged, dysregulated, as well as maladaptive reaction.⁷ Persistent inflammation is closely linked to chronic human ailments, including arthritis, cancer, atherosclerosis, allergies, as well as autoimmune diseases.⁸ Rheumatoid arthritis (RA) is ubiquitous sort of inflammatory joint ailments, featuring synovial hyperplasia and localized invasion of bone and cartilage that ultimately leads to joint destruction. It is a chronic, systemic inflammatory condition with an unclear origin. According to recent findings, individuals with RA may experience higher rates of morbidity and mortality from cardiovascular disease in addition to having a higher burden of chronic diseases than people without RA.⁹

Early on in the disease's course, a handful of joints may be afflicted, but because RA is polyarthritis, it affects multiple joints (six or more). Most peripheral joints are disease-prone; however, distal interphalangeal joints are typically spared, especially hands, feet, and knees, most severely afflicted. Another characteristic of RA is extra-articular involvement, which can include anything from dangerous vasculitis to rheumatoid nodules.¹⁰

Arthritis is more prevalent in the West than in other nations, resembling most other autoimmune ailments. Lifestyle plays significant influence, as well as dietary components may account for the geographical difference in incidence, even though the exact cause of this is not entirely understood.¹¹ Traditional RA treatments, e.g., corticosteroids, disease-modifying anti-rheumatoid drugs (DMARDs), and non-steroidal anti-inflammatory drugs (NSAIDs), are intended to lessen joint inflammation, minimize function loss, and delay advancement of joint deterioration. However, these types of therapies are rarely entirely effective, and many pharmaceutical treatments may have unintended adverse effects.¹²

Anti-inflammatory molecules are not anti-arthritic since they do not suppress responses driven by T as well as B-cells.¹³ In addition to greater dietary needs and decreased absorption of some nutrients, NSAIDs, DMARDs, and corticosteroids employed to treat disease symptoms are also linked to poor nutritional status in RA patients.¹⁴ Owing to these and other constraints, application of complementary and alternative medicine (CAM) treatments e.g., acupuncture along with herbal extracts is on rise.

Reports indicate that majority of patients with arthritis who are not pleased with their care are likely to consider CAM therapy. Many herbal remedies have been utilized historically to treat rheumatological ailments, including ailments that were similar to what is today known as RA. Salix species¹⁵, devil's claw Harpagophytum procumbens¹⁶, as well as nettle (Urtica dioica), for instance, are widely employed antirheumatic remedies in Europe. Multiple pains, encompassing joint pain, have also been reported to be treated with preparations of Zingiber officinalis¹⁷, Capsicum frutescens, Mentha piperita, Arnica montana, Curcuma longa, as well as Tanacetum parthenium¹⁸. At the molecular level, a number of herbal medicinal preparations may function as multi-component drugs, addressing multiple therapeutic targets at once.

Research conducted in vitro has demonstrated that the majority of herbal medicines taken orally have a broad mode of action. Herbal pharmaceuticals have the potential to suppress some enzymes, including elastase and hyaluronidase, that contribute to cartilage degradation, as well as cyclo-oxygenase-I or II and lipoxygenase. Thus, it would appear that some of the mechanisms of action of herbal remedies are relevant to the pathophysiology and symptoms of RA.¹⁹ Plant-based compounds with ability to control expression of pro-inflammatory have potential against arthritis encompassing elements with anti-inflammatory capabilities like flavonoids, terpenes, quinones, catechins, alkaloids, anthocyanins, along with anthoxanthins. Several of them have been extensively studied for the treatment of arthritis.²⁰

Hence authors sought to investigate anti-inflammatory as well as anti-arthritic activities of S. laurina Dalzell for leaf extracts that could serve as potential remedy for RA.

MATERIALS:

Ethanol, methanol, gelatin, dimethylsulfoxide (DMSO) were brought from S.D. Fine Chem Ltd, Mumbai. Quercetin, Folin-Ciocalteu reagent, Tetracycline HCl Acrylamide-Bisacrylamide, Tetramethylethylenediamine, Sodium dodecyl sulfate, Ammonium persulfate, Acrylamide- Bisacrylamide, Diclofenac sodium were brought Sigma-Aldrich. Mumbai. Gallic acid, Aluminium chloride, Mannitol, Tris HCl, Gelatin, egg albumen were purchased from Hi-Media Laboratories Mumbai. Every chemical, including the solvents, was of analytical and pharmaceutical grade. Throughout the investigation, double-distilled water was utilized.

METHODOLOGY:

Plant extract preparation:

Ethanolic extract:

S. laurina Dalzell leaves were collected from central Western Ghats of India location was Gund Forest, Dandeli, Uttara Kannada, in month of February, and were identified and authenticated by Taxonomy and Floristic laboratory, Karnataka Science College, Dharwad. Ref. No: KSCD/BOT/2023-24/20102 of plant deposited at same.²¹ After a week of shade drying, the leaves were blended finely. After passing through a 100-mesh screen, the powder was wrapped in polythene bags for storage. S. laurina Dalzell leaf powder was defatted with petroleum ether 60^o- 80^o, and defatted marc was extracted with ethanol via a continuous hot extraction process utilizing a Soxhlet apparatus. S. laurina Dalzell leaf extract [SLLE] was obtained by concentrating the extracts in a rotary flash evaporator (Superfit Rotary Vacuuma) and drying the residue in a vacuum desiccator over anhydrous calcium chloride. Yield of plant was recorded and was stored for further use. Extracts were subjected to preliminary phytochemical evaluation.

Aqueous extract:

S. laurina Dalzell leaf powder was macerated with chloroform water I.P. Rotary flash evaporator (Superfit Rotary Vacuuma) was used to concentrate the mixture under reduced pressure after it had been filtered through muslin cloth to obtain crude extract and finally residue was dried in vacuum desiccator over anhydrous calcium chloride to yield aqueous S. laurina Dalzell leaf extract [SLLA]. Yield of plant was recorded and was stored for further use. Extracts were subjected to preliminary phytochemical evaluation.

Study of microscopical characters²²:

Anatomical characterization:

Leaves of S. laurina Dalzell were cut and fixed in FAA (5ml formalin + 5ml acetic acid + 90ml 70% ethanol). Specimens were scheduled to be dehydrated using graded series of Tertiary Butyl Alcohol (TBA) after 24hr fixation period. Paraffin wax (M.P. 58 – 60^oC) was added gradually to carry out infiltration of specimen until TBA reached supersaturation. Specimens were put into blocks of paraffin.

Sectioning:

Sections (10–12μm thickness) of paraffin-embedded specimens were made through rotating microtome and were waxed according to protocol. Toluidine blue was used to dye certain sections. Toluidine blue is polychromatic stain, therefore excellent staining results were accompanied by certain cytochemical responses. The dye turned cellulose walls pink, lignified cells blue, suberin dark green, mucilage violet, protein bodies blue. Temporary glycerin-mounted preparations were produced for macerated materials. Following staining, powdered components of various portions were cleaned with sodium hydroxide and mounted in glycerin. Various cell components were measured as well as examined.

Photomicrographs:

Microscopic descriptions of tissues are supplemented, when necessary, with micrographs. Nickon Labphoto 2 microscopic units were utilized to capture images at multiple magnifications. The bright field was utilized for routine observations. Polarized light and starch grains lignified cells were used to analyze crystals. When exposed to polarized light, these structures look brilliant on dark background because of their birefringent properties. Scale bars indicate magnifications of figures.

Powder microscopy:

A powered plant material sample was put on a slide, covered with glass slip, and heated gradually using a microbunsen. Chloral hydrate solution was added in a few drops. Boiling too vigorously was avoided. Slide was studied under a microscope. Drop of glycerol was added after clearing procedure was finished to avoid the mounting agent from crystallizing when it cooled.

Evaluation of physical constant:

Process of standardizing S. laurina Dalzell involves assessing physical-chemical paradigms, such as extractive values, moisture content, and ash values., following standard procedure recognized according to guidelines established by WHO and will provide us with data relating to standards of quality of selected raw drug.

Preliminary phytochemical analysis:

Ethanolic as well as aqueous leaf extract were utilized for preliminary phytochemical screening of alkaloids (Meyer’s tests), carbohydrates (Molish’s, Benedict’s as well as Fehling’s test)²⁵, saponins (froth tests) tannins (gelatin test), flavonoids (alkaline reagent along with lead acetate tests).²⁷ Standard procedures were followed for screening, including tests for phenolic compounds, steroids, triterpenoids, alkaloids, carbohydrates, glycosides, flavonoids, and saponins.²⁸

Quantitative determination of secondary metabolites:

Total phenolic content estimation:

Following preparation of 1ml aliquots of 10-100μg/ml Gallic acid, 5ml tenfold-diluted Folin Ciocalteu reagent, and 4ml 75g/l sodium carbonate solution, absorbance was measured at 765nm after 30minutes. Gallic acid standard was utilized to plot calibration curve. For SLLE and SLLA (1g/100ml), 1ml was mixed separately with same reagents, as performed for constructing calibration curve. Absorbance was measured 1hr later.

Total flavonoid content estimation:

A colorimetric assay for aluminum chloride was employed to address total flavonoid concentration. Test tubes were filled with 1ml aliquots and 1ml standard quercetin solution (10–100µg/ml), and each was then filled with 4ml distilled water along with 0.3ml 5% sodium nitrite. 0.3ml 10% aluminum chloride was put in after 5 minutes. 2ml 1M sodium hydroxide was put in during sixth minute. Lastly, distilled water was added to get the volume up to 10ml and blended well. Yellowish-orange color developed. The absorbance was ascertained employing UV-visible spectrophotometer at 510nm. A calibration curve was plotted with quercetin standard. To determine the flavonoid concentration, about 1g of SLLE and SLLA were dissolved in 100ml 90% aqueous methanol as well as allowed to react with aluminum chloride, as previously mentioned. These parallel determinations were recorded. The same volume of distilled water was used in place of 10% aluminum chloride in the blank.^29-31

In-vitro anti-inflammatory activity by gelatin zymography^32,33

Detection of MMP-2 (gelatinase A) as well as MMP-9 (gelatinase B):

Negative Control:

Supernatant of squamous cell carcinoma of buccal mucosa tissue.

Positive Control: Tetracycline HCl (10mg/ml).

To put it briefly, glass plates were washed with methanol while electrophoresis apparatus was cleaned with warm water. Plates were set. Components of the clamp were assembled using a big plate, two spacers, a tiny plate over top. Agarose gel was prepared as per standard procedure, which was subsequently then heated, poured between two plates to seal bottom, allowed to cool for 5-10 minutes. The bubbling is eliminated by carefully mixing resolving gel and pouring it between glass plates. In order to create a perfectly flat interface between resolving gel and stacking gel, 80% plates were filled with stacking gel along comb, covered with small water, and left to set for approximately 45 minutes. Excess water in setting resolving gel was poured out. After being drained off, stacking gel was given about 30 mins to solidify. Post-setting stacking gel; wells washed as well as assembled gel apparatus.

MMP Samples preparation:

Following thorough chopping of the tissue sample, 5ml Tris’s buffer was put in, the mixture was centrifuged for 15 minutes at 3000rpm. 50µl of MMP samples were combined with 50µl of extract/compound (the study sample), and the mixture was incubated for 1hour. 50µl MMP sample was employed as negative control, 50µl MMP sample + 50µl Tetracycline HCl (stored for 1hr) for positive control. Each well had 20µl of sample placed in it. Run proceeded for 15 minutes at 50V, then for 100V until bromophenol blue traveled bottom of plates. Following electrophoresis, apparatus was dismantled, gel was carefully taken off and cleansed along zymogram renaturing buffer i.e., 2.5% Triton x-100 for 1hr.

Staining:

For approximately 2hrs, Coomassie R-250 de-staining solution was employed to remove the stain after 1hr of staining along Coomassie blue R-250. Background is stained blue using Coomassie dye after staining. Lower bands were gelatinases-A (MMP-2) ~72KD, while upper bands are gelatinases-B (MMP-9) ~ 95KD.^34-35

In-vitro anti-arthritic activity

Egg albumin denaturation assay:

Reaction mixture was composed of 2ml test extract with varying concentrations (μg/ml), 2.8ml phosphate buffered saline (pH 6.4), as well as 0.2ml fresh hen's egg albumin. Control was an equivalent volume of double-distilled water. Mixtures were heated for 5minutes at 70°C after 15 minutes incubation at 37±2°C in biological oxygen demand incubator. Their absorbance was ascertained at 660nm after cooling, with a vehicle serving as the blank. Final concentrations (μg/ml) of 50–500 of diclofenac sodium was reference. Test extracts were selected to stay as close to the standard therapeutic mode as practicable. Formula to compute percentage inhibition of protein denaturation:^36-38.

Abs Control – Abs Test

Percentage inhibition = ------------------------------- X 100

Abs Control

RESULT AND DISCUSSION:

Surface features:

Leaves are simple, alternate, estipulate, Petiole: 1-1.5cm long, Lamina: 10-30 x 4-10 cm, Colour: shining dark green, Base: round, Apex: obtuse, Margin: entire, lateral nerves 10-12 pairs (Figure 1a).

Study of microscopical characters

Anatomy of leaf:

Leaf consists of thick smooth lamina and biconvex midrib, midrib has thick as well as wide adaxial hump, their semicircular abaxial part (Figure 1b) midrib is 1.4mm thick; abaxial hump is 800µm wide and abaxial midrib in 1.3mm wide, cells in adaxial hump are collenchymatous; in outer zone of abaxial midrib are collenchymatous, rest of cells are parenchymatous, along adaxial part of midrib occurs arc of transcurrent of palisade parenchyma cells (Figure 1b).

Vascular system of midrib consists of deep and wide bowl shaped strand as well as two small, less prominent accessory strands situated at free adaxial ends of bowl (Figure 1b) vascular strands are collateral, these are short parallel lines of wide, angular and thick walled xylem elements as well as abaxial layer of phloem elements, these in thick arc of sclerenchyma cells abutting phloem, small accessory strands have mall cluster of xylem elements and few phloem elements; sclerenchyma cells are also associated with phloem.

Lamina:

Lamina is 450µm thick; dorsiventral with adaxial layer of palisade mesophyll along abaxial spongy parenchyma cells zone (Figure 1c). Adaxial epidermal layer is thick and cells are squarish to circular and thick walled. Cells are 70µm thick, abaxial epidermis is thin and cells are small as well as squarish. Palisade zone made up of single columnar compact cells vertical layer. Spongy mesophyll zone in thick and includes 6 or 7 layers of lobed loosely arranged cells.

Leaf Margin:

Marginal part of lamina becomes conical, slightly reduced in thickness as well as in bent down, marginal part is 150µm thick (Figure 1d). Epidermal cells along end of lamina are thicker, wide and thick walled. This is cluster of thick-walled compact cells within leaf margin.

Figure 1: a) Lower and upper epidermis of Sageraea laurina Dalzell leaves b) Leaf T.S. through midrib c) Vascular strand of midrib d) T.S. of Lamina (ABM - Midrib Abaxial part; ADH - Midrib Adaxial part; ACS - Accessory strands; COL - Collenchyma; GP - Ground Parenchyma; La - Lamina; PH - Phloem; pp - Palisade parenchyma; SC - Sclerenchyma; VB - Vascular bundle; X - Xylem).

Cystoliths: Calcium carbonate cystoliths are very common on adaxial epidermal cells of lamina (Figure 2a) epidermal or sub epidermal cells possessing cystoliths are highly widened in order to accommodate crystals; these dilated cells are lithocysts. Cystoliths are cylindrical with chinate surface; crystals in 260µm long (Figure 2c).

Epidermal glandular trichomes: Glandular trichomes are common on adaxial epidermal layer. Gland has wide dilated epidermal cell and thick, darkly stained circular body, body is four celled. Gland is pectate type.

Epidermal Cells: Adaxial epidermal cells are apostomatic, cells are highly lobed due to wary anticlinal walls (Figure 2d) cell walls are thick as well as smooth. Abaxial epidermis is densely stomatiferous and also bears glandular trichomes (Figure 3a-3e). Epidermal cells are slightly wary and thick walled. Stomata are diacytic type (Figure 3c, d, e). These are two subsidiaries for each stoma; common walls of subsidiary cells are at right angles long axis of guard cells (Figure 3d). Guard cells are elliptical in shape, measuring 30X50µm in size. Glandular trichomes are observed in surface view. Gland consists of a short stalk cells and four-celled, circular head. Head has dense cell contents and the head measures 60µm in diameter.

Petiole: Petiole is planoconvex in sectional view (Figure 4c, 4d). It is flat on adaxial side while broadly convex on abaxial side (Figure 4c) petiole is 1.8mm in vertical plane as well as 2.4mm in horizontal plane. These is continuous intact epidermal layer of squarish cells, some of epidermal cells dilated into large litho cysts possessing cystoliths (Figure 4d) grouped tissue is divided into outer zone of collenchyma eight layers cells along inner circular compact thin-walled parenchyma cells. Vascular strand is wide as well as deep bowl shaped; collateral with xylemphloem and scleranchyma tissues occurring same radius. Xylem elements are in short parallel lines with wide gaps in trichome. Xylem elements are angular as well as thick walled, phloem is in wide arc at base of xylem strand. Along lower border of phloem occurs thick arc of sclerenhyma cells.

Venation pattern of lamina:

Venation is densely reticulate (Figure 4e, and 4f), major veins are thick as well as lateral secondary vein-islets are wide; squarish of polygonal in outline. Islets have distinct vein-boundary and vein-terminatious are present in all islets. Terminatious are unbranched long or short as well as most wary. These are also branched view terminatious which is destroyed in out line. Branched terminatious arc mostly undulate.

Powder microscopy

Cystoliths: Dilated parenchyma cells called litho cysts possessing cystoliths are seen isolated in powder (Figure 5a). Lithocysts are thin-walled cells, cystoliths are narrow at one end and thick at their opposite end, surface of cystolith is echinate. Cystoliths are seen in intact epidermal peeling (Figure 5b).

Vessel Element: Isolated vessel element is occasionally seen in powder (Figure 5.1b). Vessel elements are narrow cylindrical as well as having annular or spiral thickenings and end wall perforations (Figure 5b)

Stomata: Diacytic stomata are noted in surface view of the fragmentary epidermal peelings (Figure 5c). Epidermal cells are irregular in shape as well as their anticlinal walls are wary as well as slightly thick, guard cells are elliptical in outline. (Figure 5c, 5d) Pectate types of epidermal glandular trichomes are common on epidermis. Gland has circular outline. It is four celled and cells are circulating in arrangement (Figure 5c).

Figure 2: a) T.S. of Lamina through marginal part b) Marginal part T.S. of the leaf showing cystoliths c) T.S. of leaf showing glandular trichome d) Lithocysts and cystolith seen in surface view of leaf (ABE - Abaxial epidermis; ADE - Adaxial epidermis; CU - Cuticle; LM - Leaf Margin; PM - Palisade Mesophyll; SM - Spongy Mesophyll)

Figure 3: a) Adaxial epidermis in paradermal section b) Abaxial epidermal layer showing stomatal morphology c) Appearance of Diacytic stomata enlarged EC - Epidermal cells; GC - Guard cells; SC - Subsidiary cells; ST – Stomata d and e) Appearance of Stomata as seen in surface viewing paradermal sections.

Figure 4: a) Epidermal peeling of lamina showing stomata and glandular trichome b) Structure of Glandular trichome in surface view c) T.S. of petiole-entire view d) COL - collenchymas; SEL - Sclerenchyma cells; CY - Cystolith; EP - Epidermis SCl - Sclerenchyma; X – Xylem; Pa - Parenchyma; PH – Phloem; R - Ridge; e) and f) Venation pattern of the lamina

Figure 5: a) Vessel elements with lateral wall thickenings b) Parenchymatous lithocyst possessing a cystolith (CL- cystoliths, LC – Lithocyst, PE- Phloem elements, SCT-semicircular trichomes, VE-vessel elements) c) Paradermal sectioning of abaxial epidermis showing stomata and glandular trichome d) Epidermal layer showing the cystolith CY - Cystolith EC- Epidermal cells, ST – Stomata

Preliminary phytochemical analysis:

Phytochemical screening has potential to facilitate drug discovery and development by identifying bioactive chemicals found in medicinal plants. A medicinal plant's phytoconstituents, either alone or in combination, contribute to its therapeutic value.

Variety of biological actions are exhibited by significant phytochemicals. Nature is unique source of structures with high phytochemical diversity. Phytochemical identification allows for prediction of plant's pharmacological activity. Phytochemical investigation of SLLE confirmed existence of carbohydrates, flavonoids, glycosides, steroids, alkaloids, tannins along phenolic compounds as well as existence of carbohydrates, flavonoids, saponin glycosides tannins along phenolic compounds in SLLA. Extraction yield percentage was 8.73% in SLLE and it was 4.28% in SLLA. Loss on drying was 6.94g. Different ash values are vital to decide purity of medication i.e., occurrence or nonappearance of foreign inorganic matter. Table 1 displays proximate values of S. laurina Dalzell leaves.

Table 1: Proximate values of S. laurina Dalzell leaves:

Sr. No.	Physicochemical Parameters		Determined Average values (%) (n=3)
1	Loss on Drying	Moisture content	6.94
2	Extractive Values	Alcohol soluble extractive	9.24
2	Extractive Values	Water soluble extractive	5.65
3	Ash Values	Total ash	1.28
		Acid insoluble ash	0.43
		Water soluble ash	0.89
		Sulphated ash	1.05

Quantitative determination of secondary metabolites

Estimation of total phenolic content:

Extracts from leaves of S. laurina Dalzell are rich in phenolic as well as flavonoid components. Because of their redox characteristics, phenolic substances can function as antioxidants.³⁹ Total phenolic content was ascertained using Folin-Ciocalteau technique. Gallic acid equivalents are used to express phenolic content, which was computed by applying regression equation of standard plot (y = 0.0045x + 0.0028, R² = 0.9991). Total Phenol content present in SLLE and SLLA were 93.27mg GAE/g and 60.45mg GAE/g respectively.

Estimation of total flavonoid content:

Occupancy of free OH groups, especially 3-OH, is necessary for antioxidant efficacy of plant secondary metabolites known as flavonoids, which include flavones, flavanols, along condensed tannins. They are synthesized by acetate/malonate and shikimate/ phenylpropanoid pathways. They are composed of molecules with common C6-C3-C6 skeleton, in which two benzene rings are coupled by virtue of three-carbon bridge, uniquely forming phenylchromane configuration.³⁸Aluminum chloride technique was employed for assessing total flavonoid content. Flavonoid content, which is reported as quercetin equivalents, was determined by virtue of regression equation of standard plot (y = 0.0038x + 0.0001, R² = 0.9991). Total flavonoid content present in SLLE as well as SLLA are 86.21mg QUE/g and 52.29 mg QUE/g respectively.

In-vitro anti-inflammatory activity:

Alcoholic and aqueous leaf extracts of S. laurina Dalzell were assessed for anti-inflammatory efficacy against MMP-2 as well as MMP-9 by virtue of gelatin zymography using tetracycline as reference drug. A sensitive and effective technique for identifying proteolytic enzymes that may be degrading gelatin from a variety of biological sources is gelatin zymography. Its powerful gelatin-degrading activity makes it valuable for evaluating the matrix metalloproteinase family, encompassing MMP-2 as well as MMP-9. Neutrophils, macrophages, fibroblasts, endothelial cells, along epithelial cells are examples of activated inflammatory cells producing MMPs.⁴⁰

MMPs are enzymes that degrade extracellular matrix along basement membrane, control influx of inflammatory cells, and hence aids the remodeling of tissue.⁴¹ Gelatinase family of MMPs, comprising MMP-9 and MMP-2, is crucial in turnover along with degradation of extracellular matrix proteins during cellular recruitment in inflammation.^42,43 MMP-9 is significantly induced in airway epithelial cells by virtue of inflammatory cytokines, specifically TNF-α, while MMP-2 is produced in various cell types.^44-47 Consequently, corresponding expression extents of MMP-9 were normalized to MMP-2 values. This technique measures the efficacy of gelatinases, particularly MMP-2 along MMP-9, while they are with their pro-domains and demand denaturation to get activated. Following Coomassie blue staining, it was then visible as single or double white bands (pro along active forms) on gelatin zymograms.

The percentage bands of MMP-2 along MMP-9 that were found for every sample screened by virtue of gelatin zymogram using a gel electrophoresis, crucially determines in vitro anti-inflammatory outcomes.^46-49 It was observed that SLLE and SLLA were able to act as agents that assess protein enzymatic activity either in situ or by virtue of separating them by electrophoresis. Proteolysis sites were visible as translucent bands over dark blue background when gel was stained by Coomassie blue. Where gelatin is degenerated, Coomassie stain causes the background to turn blue.

The appearance of white bands indicates existence of gelatinases. Upper bands are MMP-9 which runs at 95 KB and lower bands are MMP-2 (Figure 6). Percentage inhibitions of SLLE have shown comparably good activity as compared to SLLA. Percentage of inhibition for anti-inflammatory potential of SLLE against MMP-2 as well as MMP-9 were found to be 62% and 56% respectively and SLLA against MMP-2 and MMP9 were found to be 34% and 32% respectively. Said investigation specifies that SLLE and SLLA notably diminish MMP-9 activity in IT-stimulated BEAS-2B cells. Results for in-vitro anti-inflammatory activity of SLLE along SLLA are tabulated in Table 2.

Figure 6: Bands showing anti- inflammatory activity after electrophoresis

Table 2: In-vitro anti-inflammatory activity of SLLE and SLLA by Gelatin zymography method.

Sr. No.	Name of the Extracts/ drugs	% Bands of MMP		% Inhibition of MMP
Sr. No.	Name of the Extracts/ drugs	MMP 2	MMP 9	MMP 2	MMP 9
1	Negative Control (Supernatant of Squamous cell carcinoma)	100	100	00	00
2	SLLE	38	44	62	56
3	SLLA	66	68	34	32
4	Positive Control (Tetracycline HCL)	00	00	100	100

In-vitro anti-arthritic activity:

Inhibition of egg albumen protein denaturation:

One of well-established root cause of inflammatory and arthritic disorders is tissue proteins denaturation. Denaturation of proteins may be cause of autoantigen production in some arthritic conditions. Therefore, it would be beneficial to discover agents that can stop protein denaturation to produce anti-arthritic drugs. Anti-arthritic activity of alcoholic and aqueous leaf extracts of S. laurina Dalzell were studied by using inhibition of egg albumin protein denaturation method and results are illustrated in Table 3 and Figure 7.

It was observed that SLLE and SLLA were able to act as agents that prevent protein denaturation thereby indicating itself to possess anti-arthritic activity which was exhibited by increase in absorbance compared with control. It was noted that at concentration 500µg/ml, percentage inhibition of SLLE and SLLA was 36.23-74.14% and 26.10-60.30% with IC50 value 17.23µg/ml and 31.19µg/ml respectively, standard drug diclofenac sodium at same concentration was found to be 45.08 to 84.53% with IC50 value 8.24µg/ml.

Table 3: In-vitro anti-arthritic potential of SLLE and SLLA by virtue of egg albumin protein denaturation approach.

Conc (µg/ml)	Mean % Inhibition
Conc (µg/ml)	SLLE	SLLA	Diclofenac sodium
50	36.23	26.10	45.08
100	46.68	38.23	52.28
200	54.10	42.53	61.24
300	60.43	50.10	70.14
400	70.02	56.02	79.43
500	74.14	60.30	84.53
IC₅₀	17.23	31.19	8.24

Percentage of Inhibition, n=3. All values are significant at P<0.001

Figure 7: In-vitro anti-arthritic activity of SLLE and SLLA by egg albumin protein denaturation method.

CONCLUSION:

Findings of current research work suggest that presence of secondary metabolites e.g., flavonoids, steroids, alkaloids, saponin glycosides, tannins, as well as phenolic compounds all of which have been repeatedly noted to hold anti-inflammatory as well as anti-arthritic properties in S. laurina Dalzell leaf extracts have contributed to inhibition of various endogenous inflammatory mediators, joint pain transmission, and mediators. S. laurina Dalzell ethanolic extract exhibited maximum anti-inflammatory and anti-arthritic activities as compared to aqueous extract and hence aids as remedy of RA. This offered large window of opportunity for development of S. laurina Dalzell's broad-spectrum application in herbal medicine and as a foundation for developing new, highly effective medications against inflammatory arthritis.

CONFLICT OF INTEREST:

Conflict of interest declared none.

ABBREVIATION:

Abbreviation	Meaning
S. laurina Dalzell	Sageraea laurina Dalzell
SLLE	S. laurina Dalzell leaf ethanolic extract
SLLA	S. laurina Dalzell leaf aqueous extract
TBA	Tertiary butyl alcohol
hr	Hour
MMP-2	Gelatinase A
MMP-9	Gelatinase B
^oC	Degree centigrade
DMARDs,	Disease-modifying anti-rheumatoid drugs
NSAIDs,	Non-steroidal anti-inflammatory drugs
CAM	Complementary and alternative medicine

REFERENCE:

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Received on 01.03.2024 Modified on 05.04.2024

Res. J. Pharmacognosy and Phytochem. 2024; 16(2):59-68.

DOI: 10.52711/0975-4385.2024.00012

Surface features:

Leaves are simple, alternate, estipulate, Petiole: 1-1.5cm long, Lamina: 10-30 x 4-10 cm, Colour: shining dark green, Base: round, Apex: obtuse, Margin: entire, lateral nerves 10-12 pairs (Figure 1a).

Study of microscopical characters

Table 1: Proximate values of S. laurina Dalzell leaves:

Sr. No.

Physicochemical Parameters

Determined Average values (%) (n=3)

1

Loss on Drying

Moisture content

6.94

2

Extractive Values

Alcohol soluble extractive

9.24

Water soluble extractive

5.65

3

Ash Values

Total ash

1.28

Acid insoluble ash

0.43

Water soluble ash

0.89

Sulphated ash

1.05