PHARMACOGNOSTICAL AND PHYTOPHARMACOLOGICAL INSIGHTS INTO PIPER LONGUM L. LEAVES WITH GC–MS AND HPTLC FINGERPRINTING

PHARMACOGNOSTICAL AND PHYTOPHARMACOLOGICAL INSIGHTS INTO PIPER LONGUM L. LEAVES WITH GC–MS AND HPTLC FINGERPRINTING

S. A. Abith *, P. Meena Prabha and B. Sangameswaran

Department of Pharmacognosy, S. S. M. College of Pharmacy, Chinniyampalayam, Erode, Tamil Nadu, India.

ABSTRACT: Background: Piper longum L. (long pepper) is a well-known Ayurvedic medicinal plant used for metabolic, inflammatory, and infectious disorders, yet its leaves remain relatively underexplored despite their traditional relevance. Objective: This study aimed to investigate the pharmacognostic, phytochemical, and pharmacological properties of Piper longum leaves, focusing on their antidiabetic, anti-inflammatory, antibacterial, and antifungal activities. Methods: Leaves of P. longum were collected, authenticated, and evaluated through pharmacognostic and physicochemical analyses. Phytochemical screening identified key secondary metabolites, while GC–MS and HPTLC provided compound profiling and fingerprinting. In-vitro assays assessed antidiabetic activity (α-amylase and α-glucosidase inhibition), anti-inflammatory effects (albumin denaturation and HRBC membrane stabilization), and antimicrobial activity against selected bacterial and fungal pathogens. Results: Pharmacognostic analysis confirmed distinct diagnostic features of the leaves, while phytochemical screening identified alkaloids, flavonoids, tannins, terpenoids, saponins, phenolics, and glycosides. GC–MS revealed key constituents including asarone, β-caryophyllene, phytol derivatives, and piperine-like alkaloids and HPTLC provided reproducible chemical fingerprints. The extract showed strong, dose-dependent α-amylase and α-glucosidase inhibition, notable anti-inflammatory effects comparable to aspirin, and significant antimicrobial activity, with highest inhibition against P. acnes and C. albicans, in some cases surpassing standard drugs. Conclusion: These findings validate Piper longum leaves as a pharmacologically versatile source of bioactive compounds with broad therapeutic potential. Their combined antidiabetic, anti-inflammatory, and antimicrobial activities, supported by strong phytochemical evidence, justify traditional uses and highlight their promise for development into standardized herbal formulations and modern phytopharmaceuticals.

Keywords: Piper longum, Pharmacognosy, GC–MS, HPTLC, Antidiabetic, Anti-inflammatory, Antibacterial, Antifungal, Piperine

INTRODUCTION: Herbal medicine utilizes plant-derived and natural substances for therapeutic purposes, although many traditional claims remain insufficiently validated ^{1, 2}.

Their efficacy is attributed to bioactive constituents such as phenolics, flavonoids, alkaloids, terpenoids, and saponins, which confer diverse pharmacological activities ^3-5.

Piper longum L. (long pepper), an aromatic climber native to South and Southeast Asia, contains piperine, lignans, and essential oils that support its extensive traditional use in managing digestive, respiratory, metabolic, and microbial disorders ^6-9. Historical records trace its use to ancient Greek and Roman civilizations, with the term “pepper” derived from the Tamil pippali ¹⁰. In modern contexts, rising global burdens of diabetes, chronic inflammation, and microbial resistance highlight the need for safer, multi-targeted alternatives to synthetic drugs. Plant-based agents such as Gymnema sylvestre, curcumin, and boswellic acids exemplify this potential, while increasing fungal (C. albicans) and bacterial resistance underscores the importance of exploring phytochemicals as novel antimicrobial candidates ^11-17.

FIG. 1: PIPER LONGUM PLANT

MATERIALS AND METHODS: Fresh leaves of Piper longum L. were collected from the Western Ghats region of Tamil Nadu and taxonomically confirmed by Dr. P. Radha (CCRS). A voucher specimen was archived in the departmental herbarium for future verification. Pharmacognostic analysis began with macroscopic characterization, documenting leaf color, texture, odor, taste, size, shape, and venation, along with photographic imaging for diagnostic clarity ¹⁸. Powdered shade-dried leaves were examined microscopically using histochemical reagents specific for lignin, cellulose, and starch ¹⁹. Fresh leaf samples were cleared in NaOH, sectioned, and mounted in glycerin for quantitative microscopy, enabling measurement of stomatal number, stomatal index, epidermal cell density, and palisade ratio ²⁰. Additional morphological descriptors leaf margin, base, apex, and venation pattern were also recorded ²¹.

For detailed anatomical study, plant tissues were fixed in FAA, dehydrated through a tertiary butyl alcohol series, and embedded in paraffin following the classical procedure of Sass (1940) ²². Thin transverse sections (10–12 µm) were cut, dewaxed, and stained with toluidine blue, safranin, fast green, or IKI to distinguish structural components and localize cell wall biopolymers such as lignin, cellulose, suberin, mucilage, and starch ²³. Epidermal peels and paradermal sections were prepared using 5% NaOH or Jeffrey’s fluid to observe stomatal types, venation architecture, and trichome morphology ²⁴. Photomicrographs were captured with a Nikon LabPhoto-2 microscope under bright-field and polarized light conditions ²⁵.

Physicochemical constants were assessed using standard protocols. Moisture content was measured as loss on drying at 105 °C ²⁶. Total ash was obtained after incineration at 450–600 °C ²⁷, while acid-insoluble ash and water-soluble ash were quantified through treatment with 6 N HCl and boiling water, respectively ^{28, 29}. Alcohol-soluble and water-soluble extractive values were determined using 95% ethanol and distilled water extraction procedures ^{30, 31}. Volatile oil content was estimated by hydrodistillation using a Clevenger apparatus ³². The swelling and foaming indices were measured to assess mucilage and saponin content ^{33, 34}, and pH was recorded with a calibrated digital pH meter ³⁵.

For phytochemical screening, the air-dried powdered leaves were subjected to Soxhlet extraction with ethanol. The resulting extract was concentrated under reduced pressure, and attributes such as yield, consistency, and color were documented ^{36, 37}. Standard qualitative assays were used to test for major secondary metabolites including phenolics, tannins, alkaloids, flavonoids, terpenoids, saponins, glycosides, steroids, carbohydrates, proteins, and quinones ³⁸.

Chemical profiling was performed using GC–MS equipped with a DB-5 MS capillary column. The instrument was operated with helium as carrier gas (1 mL/min), injector temperature 250 °C, linear oven ramp from 60–280 °C, EI ionization at 70 eV, and a mass scan range of 50–600 m/z ³⁹. HPTLC fingerprinting utilized silica gel 60 F₂₅₄ plates and a mobile phase of toluene–ethyl acetate–formic acid (7:2:1). Samples were applied with a CAMAG Linomat-5 system, scanned at 254 and 366 nm, and derivatized with anisaldehyde–sulphuric acid for visualization ⁴⁰.

The pharmacological activities of the extract were examined through a series of in-vitro assays. Antidiabetic activity was evaluated via α-amylase inhibition by quantifying DNS-reactive reducing sugars at 540 nm after incubation with starch substrate (250–1000 µg/mL extract) ⁴¹. α-Glucosidase inhibition was determined by measuring glucose release during sucrose hydrolysis using a chromogenic reagent at 510 nm ⁴². Anti-inflammatory potential was examined through inhibition of heat-induced albumin denaturation at 660 nm, with acetyl salicylic acid as reference ⁴³, and by HRBC membrane stabilization based on percent hemolysis inhibition at 560 nm ⁴⁴.

Antifungal effects against Candida albicans and Aspergillus niger were assessed using agar well and disc diffusion techniques on PDA plates, employing Amphotericin B as the standard drug ⁴⁵. Antibacterial activity against Staphylococcus aureus, Propionibacterium acnes, Escherichia coli, and Enterococcus faecalis was tested using agar well diffusion on nutrient agar, with gentamicin as the positive control. Zones of inhibition were recorded after 24 h incubation at 37 °C and analyzed using GraphPad Prism 6.0 software ⁴⁶.

RESULTS AND DISCUSSION:

Macroscopy of Leaf: The leaves are simple, alternate, and entire, borne on distinct petioles, and exhibit a lanceolate to ovate-lanceolate outline with a cordate base and acute apex. They possess a thin, membranous, glabrous surface with clearly visible basal ribs. The lamina typically measures 4–7 × 2–3 cm, while the petiole extends 1–2 cm in length.

TABLE 1: ORGANOLEPTIC CHARACTERS OF P. LONGUM L. (LEAF)

Microscopy of Leaf: The transverse section of the petiole exhibits a reniform outline, characterized by a convex abaxial and a concave adaxial surface. The epidermis is uniseriate and covered by a thin cuticle, bearing numerous unicellular to multicellular uniseriate nonglandular trichomes. Beneath the epidermis, the hypodermis comprises 5–6 continuous layers of collenchyma along the adaxial surface, whereas the abaxial and lateral regions are interrupted by parenchymatous cells. The outer cortex consists of 3–4 layers of chlorenchyma on the upper side and small parenchyma cells below, while the inner cortex and ground tissue are composed of large, thin-walled parenchyma. The vascular system is arranged in a crescent-shaped arc containing 8–10 conjoint, collateral, and closed vascular bundles, with xylem elements positioned adaxially (mesarch configuration) and phloem oriented toward the periphery.

The midrib, in transverse section, appears biconvex with an angular adaxial surface and a semicircular abaxial surface. A uniseriate epidermis with a delicate cuticle covers both surfaces, and unicellular to 2–4-celled uniseriate nonglandular trichomes are confined to the abaxial side. The hypodermis consists of 5–6 layers of collenchyma beneath both the upper ridge and the lower epidermis. Two layers of thin-walled parenchyma occur on either side of the adaxial hypodermis and extend into the lamina as enlarged parenchyma cells. Beneath the hypodermis lie 3–4 layers of chlorenchyma, which merge into the mesophyll. A single large conjoint, collateral, closed vascular bundle is centrally positioned within the ground tissue, which is composed of broad parenchyma cells, with xylem oriented adaxially and phloem located toward the abaxial side. Epidermal peel preparations revealed a hypostomatic leaf surface with abundant anomocytic stomata, each guard cell pair surrounded by four uniformly sized subsidiary cells. Both upper and lower epidermal layers consist of large, polygonal parenchymatous cells.

FIG. 2: TRANSVERSE SECTION OF PIPER LONGUM LEAF

Powder Microscopy: Microscopic analysis of Piper longum leaves demonstrated characteristic cellular features, including polygonal epidermal cells, anomocytic stomata, deposits of oleoresins, calcium oxalate crystals, stone cells, fibrosclereids, and fragments of xylem vessels exhibiting pits and scalariform thickenings, along with dispersed mesophyll tissue.

FIG. 3: XYLEM VESSEL WITH SCALARIFORM THICKENING AND XYLEM VESSEL WITH PITTED AND SCALARIFORM THICKENING

FIG. 4: BROWN CONTENT/OLEORESIN CONTENT

FIG. 5: SPIRAL THICKENING OF XYLEM VESSEL

FIG. 6: STONE CELLS                                   

FIG. 7: FIBROSCLEREID

FIG. 8: EPIDERMAL CELLS                       

FIG. 9: TETRACYTIC STOMATA

FIG. 10: UPPER EPIDERMIS                       

FIG. 11: LOWER EPIDERMIS

FIG. 12: VEIN TERMINATIONS                           

FIG. 13: VEIN ISLET

TABLE 2: QUANTITATIVE MICROSCOPY

TABLE 3: PHYSICO- CHEMICAL ANALYSIS

TABLE 4: ETHANOLIC EXTRACTION RESULTS OF PIPER LONGUM L. LEAVES

 TABLE 5: PRELIMINARY PHYTOCHEMICAL ANALYSIS (+ PRESENT; - ABSENCE)

HPTLC Analysis of Piper longum Extract: High-performance thin-layer chromatography (HPTLC) of the ethanolic leaf extract revealed a chemically diverse profile under both UV (254 and 366 nm) and post-derivatization conditions.

Distinct chromatographic bands were observed within Rf ranges of 0.08–0.72 (254 nm), 0.08–0.71 (366 nm) and exhibited purple and green coloration after derivatization. Prominent, reproducible bands at Rf 0.62–0.72 corresponded to marker alkaloids such as piperine, validating both chemical diversity and method reliability.

Densitometric analysis at 254 nm (Track 1, 5 µL) displayed ten peaks, with the major peak at Rf ≈ 0.58 (33.68% area), while moderate peaks at Rf 0.68–0.81 and minor peaks below Rf 0.35 indicated additional secondary metabolites. Track 2 (7 µL) exhibited ten peaks, with the dominant peak at Rf 0.58 (33.68%) identified as piperine, and secondary peaks at Rf 0.74 (14.97%) and Rf 0.81 (12.40%) likely representing related amide alkaloids such as piperlongumine or pipernonaline; minor peaks (Rf 0.05–0.27) suggested additional constituents.

At 366 nm, Track 1 (5 µL) showed nine peaks across Rf 0.02–0.86, with the major peak at Rf 0.68 (37.05%) and high-intensity peaks at Rf 0.08 and 0.58 (16.29% each), together accounting for ~70% of total area. Track 2 (7 µL) revealed nine peaks, dominated by Rf 0.68 (41.57%), followed by Rf 0.57 (18.94%) and Rf 0.07 (12.78%), with additional minor peaks confirming a reproducible chemical profile.

At 575 nm, Track 1 (5 µL) displayed 11 peaks, with major compounds at Rf 0.61 (28.14%) and Rf 0.86 (24.85%), and moderate peaks at Rf 0.44 (13.76%) and Rf 0.71 (8.37%). Track 2 (7 µL) showed 12 resolved peaks, with prominent peaks at Rf 0.59 (28.13%) and Rf 0.86 (25.31%), moderate peaks at Rf 0.42 (13.44%), 0.76 (6.58%), and 0.51 (6.10%), and minor peaks (<5%), reflecting a highly diverse and well-resolved phytochemical profile.

FIG. 14: HPTLC UNDER UV SHORT                          

FIG. 15: HPTLC UNDER UV LONG                  

FIG. 16: HPTLC UNDER WHITE LIGHT AFTER DERIVATISATION

FIG. 17: HPTLC TRACK 1- 5 µL, 254 NM

TABLE 6: HPTLC DENSITOGRAM PROFILE (TRACK 1 – 5, µL254 NM)

FIG. 18: HPTLC TRACK 2 – 7 µL, 254 NM

TABLE 7: : HPTLC DENSITOGRAM PROFILE (TRACK 2 – 7 μL, 254 NM)

FIG. 19: HPTLC TRACK 1 – 5 µL, 366 NM

TABLE 8: HPTLC DENSITOGRAM PROFILE (TRACK 1 – 5 µL, 366 NM)

FIG. 20: HPTLC TRACK 2 – 7 µL, 366 NM

TABLE 9: HPTLC DENSITOGRAM PROFILE (TRACK 2 – 7 µL, 366 NM)

FIG. 21: HPTLC TRACK 1- 5 µL, 575 NM

TABLE 10: HPTLC DENSITOGRAM PROFILE (TRACK 1 – 5 µL, 575 NM)

FIG. 22: HPTLC TRACK 2 – 7 µL, 575 NM

TABLE 11: HPTLC DENSITOGRAM PROFILE (TRACK 2 – 7 µL, 575 NM)

GC–MS (GAS Chromatography–Mass Spectrometry) Analysis: GC–MS analysis of Piper longum leaf extract revealed a chemically diverse profile, with major constituents including alkaloids (notably piperine), terpenoids (β-caryophyllene, humulene, α-pinene, limonene), phenolics, esters, fatty acids, and hydrocarbons. The dominant compound was 1,3-bis-(2-cyclopropyl,2-methylcyclopropyl)-but-2-en-1-one (31.93%), followed by 3,7,11,15-tetramethyl-2-hexadecen-1-ol (4.39%), asarone (2.62%), benzene,1,2,3-trimethoxy-5-(2-propenyl)- (3.32%), and 9-phenanthrenemethyl 2,6-dimethylbenzoate (3.37%). Minor peaks, including cyclotrisiloxane derivatives and esters, reflect a complex array of phenylpropanoids, terpenoids, heterocycles, esters, amides, and siloxanes. The major compounds likely underlie the observed antidiabetic, anti-inflammatory, antimicrobial, antioxidant, and neuroprotective activities, while minor constituents may act synergistically; alkaloids contribute primarily to antidiabetic and antimicrobial effects, terpenoids to anti-inflammatory and antimicrobial actions, and phenolics to antioxidant and anti-inflammatory potential.

FIG. 23: CHROMATOGRAM - GCMS

TABLE 12: RESULT – GCMS

In-vitro Anti Diabetic Activity:

Alpha Amylase Inhibitory Assay:

TABLE 13: OD VALUE AND PERCENTAGE INHIBITION OF STANDARD (ACARBOSE) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) FOR ALPHA AMYLASE INHIBITORY ASSAY

IC50 Value-Standard: 220.059 µg/mL (Calculated using ED50 Plus V 1.0 Software). IC50 Value-sample: 940.068 µl/mL (Calculated using ED50 Plus V 1.0 Software).

FIG. 24: LINE GRAPH COMPARISON OF PERCENTAGE INHIBITION BETWEEN THE STANDARD (ACARBOSE) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) OF ALPHA AMYLASE INHIBITORY ASSAY

FIG. 25: BAR GRAPH COMPARISON OF PERCENTAGE INHIBITION BETWEEN THE STANDARD (ACARBOSE) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) OF ALPHA AMYLASE INHIBITORY ASSAY

Alpha Glucosidase Inhibitory Assay:

TABLE 14: OD VALUE AND PERCENTAGE INHIBITION OF STANDARD (ACARBOSE) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) FOR ALPHA GLUCOSIDASE INHIBITORY ASSAY

IC₅₀ Value –Standard –229.45 µg/mL (Calculated using ED₅₀ PLUS V1.0 Software). IC₅₀ Value –sample –717.393 µl/mL (Calculated using ED₅₀ Plus V1.0 Software).

FIG. 26: LINE GRAPH COMPARISON OF PERCENTAGE INHIBITION BETWEEN THE STANDARD (ACARBOSE) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) OF ALPHA GLUCOSIDASE INHIBITORY ASSAY

FIG. 27: BAR GRAPH COMPARISON OF PERCENTAGE INHIBITION BETWEEN THE STANDARD (ACARBOSE) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) OF ALPHA GLUCOSIDASE INHIBITORY ASSAY

In-vitro Anti Inflammatory Activity:

Inhibition of Albumin Denaturation:

TABLE 15: OD VALUE AND PERCENTAGE INHIBITION OF STANDARD (ASPIRIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) FOR INHIBITION OF ALBUMIN DENATURATION ASSAY

IC50 Value of standard: 84.2 μg/ml, IC50 Value of tested sample: 136.8 μg/ml.

FIG. 28: LINE GRAPH COMPARISON OF PERCENTAGE INHIBITION BETWEEN THE STANDARD (ASPIRIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) OF INHIBITION OF ALBUMIN DENATURATION ASSAY

FIG. 29: BAR GRAPH COMPARISON OF PERCENTAGE INHIBITION BETWEEN THE STANDARD (ASPIRIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) OF INHIBITION OF ALBUMIN DENATURATION ASSAY

HRBC Membrane Stabilization Method:

TABLE 16: OD VALUE, PERCENTAGE OF HEMOLYSIS AND PERCENTAGE OF PROTECTION OF STANDARD (ASPIRIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) FOR HRBC MEMBRANE STABILIZATION METHOD

IC50 Value of standard: 82 μg/ml, IC50 Value of tested sample: 140.2 μg/ml

FIG. 30: LINE GRAPH COMPARISON OF PERCENTAGE PROTECTION AND PERCENTAGE HEMOLYSIS OF STANDARD (ASPIRIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) HRBC MEMBRANE STABILIZATION METHOD

FIG. 31: BAR GRAPH COMPARISON OF PERCENTAGE PROTECTION AND PERCENTAGE HEMOLYSIS OF STANDARD (ASPIRIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) IN HRBC MEMBRANE STABILIZATION METHOD

In-vitro Anti Microbial Activity:

Anti Fungal Activity:

Agar Well Diffusion Method:

TABLE 17: MEANS ± SD OF ZONE OF INHIBITION OBTAINED BY POSITIVE CONTROL (AMPHOTERICIN B) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) AGAINST CANDIDA ALBICANS AND ASPERGILLUS NIGER

SD – Standard Deviation, *Significance - p< 0.05

FIG. 32: LINE GRAPH COMPARISON BETWEEN ZONE OF INHIBITION OBTAINED BY POSITIVE CONTROL (AMPHOTERICIN B) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) AGAINST CANDIDA ALBICANS AND ASPERGILLUS NIGER

FIG. 33: BAR GRAPH COMPARISON BETWEEN ZONE OF INHIBITION OBTAINED BY POSITIVE CONTROL (AMPHOTERICIN B) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) AGAINST CANDIDA ALBICANS AND ASPERGILLUS NIGER

Anti Bacterial:

Agar Well Diffusion Method:

TABLE 18: MEANS ± SD OF ZONE OF INHIBITION OBTAINED BY POSITIVE CONTROL (GENTAMICIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) AGAINST TEST ORGANISMS

SD – Standard Deviation, *Significance - p< 0.05.

FIG. 34: LINE GRAPH COMPARISON BETWEEN ZONE OF INHIBITION OBTAINED BY POSITIVE CONTROL (GENTAMICIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) AGAINST S. AUREUS, P. ACNES, E. COLI AND E. FAECALIS

FIG. 35: BAR GRAPH COMPARISON BETWEEN ZONE OF INHIBITION OBTAINED BY POSITIVE CONTROL (GENTAMICIN) AND SAMPLE (P. LONGUM L. LEAF ETHANOLIC EXTRACT) AGAINST S. AUREUS, P. ACNES, E. COLI AND E. FAECALIS

DISCUSSION: The ethanolic extract of Piper longum leaves exhibited potent inhibitory activity against α-amylase and α-glucosidase, indicating its potential to modulate postprandial hyperglycemia. Dual inhibition at both early (α-amylase) and late (α-glucosidase) stages of carbohydrate digestion suggests a synergistic mechanism for effective glycemic control. Bioactive constituents, including piperine, flavonoids, and tannins, may mediate these effects through competitive or noncompetitive enzyme inhibition and conformational modulation. The anti-inflammatory activity of the extract is likely associated with stabilization of cellular membranes and prevention of protein denaturation, thereby limiting the release of inflammatory mediators.

Piperine has been reported to downregulate NF-κB, COX-2, and TNF-α signaling, while flavonoids and tannins contribute antioxidant activity and enzyme inhibition, enhancing membrane protection.

The extract also demonstrated broad-spectrum antimicrobial efficacy. Compounds such as piperine, piperlongumine, and β-caryophyllene may disrupt microbial membranes, interfere with nucleic acid synthesis, and induce oxidative stress, inhibiting growth of both Gram-positive and Gram-negative bacteria as well as fungi. These results underscore the therapeutic versatility of P. longum leaves, supporting their traditional use and potential development as a natural antimicrobial and anti-inflammatory agent.

CONCLUSION: This study comprehensively evaluated Piper longum L. leaves through pharmacognostic, phytochemical, and pharmacological analyses. Key diagnostic features and chemical markers, including piperine and related alkaloids, ensure reliable authentication and standardization. The ethanolic extract exhibited potent antidiabetic, anti-inflammatory, and broad-spectrum antimicrobial activities, supporting its traditional use. The multifaceted pharmacological effects, combined with piperine’s bioavailability-enhancing properties, highlight P. longum leaves as a promising candidate for development into multi-targeted, plant-based therapeutics.

ACKNOWLEDGEMENTS: Nil

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. WHO Traditional Medicine Strategy 2014–2023. Geneva: World Health Organization 2013.
2. Ekor M: The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol 2014; 4: 177.
3. Panche AN, Diwan AD and Chandra SR: Flavonoids: an overview. J Nutr Sci 2016; 5: 47.
4. Kumar S and Pandey AK: Chemistry and biological activities of flavonoids: an overview. Sci World J 2013; 2013: 162750.
5. Gupta SC, Prasad S, Kim JH, Patchva S, Webb LJ, Priyadarsini IK and Aggarwal BB: Multitargeting by curcumin as revealed by molecular interaction studies. Nat Prod Rep 2011; 28(12): 1937–55.
6. Nadkarni KM: Indian Materia Medica 3rd ed. Bombay: Popular Prakashan 1976; 1.
7. Warrier PK, Nambiar VPK and Ramankutty C: Indian Medicinal Plants: A Compendium of 500 Species. Madras: Orient Longman 1995; 4.
8. Srivastava A, Shukla YN and Kumar S: Chemistry and pharmacology of Piper longum L. Indian Drugs 1999; 6(6): 373–8.
9. Khushbu C, Roshni S, Anar P, Carol M and Mayuree P: Piper longum: Traditional uses, phytochemistry and pharmacological activities—a review. Int J Curr Pharm Res 2011; 3(4): 1–11.
10. Parthasarathy VA, Chempakam B and Zachariah TJ: Chemistry of Spices. Wallingford: CABI Publishing; 2008.
11. Patel DK, Prasad SK, Kumar R, Hemalatha S. An overview on antidiabetic medicinal plants having insulin mimetic property. Asian Pac J Trop Biomed 2012; 2(4): 320–30.
12. Modak M, Dixit P, Londhe J, Ghaskadbi S, Paul A and Devasagayam TPA: Indian herbs and herbal drugs used for the treatment of diabetes. J Clin Biochem Nutr 2007; 40(3): 163–73.
13. Calixto JB, Campos MM, Otuki MF and Santos ARS: Anti-inflammatory compounds of plant origin. Part II. Modulation of pro-inflammatory cytokines, chemokines and adhesion molecules. Planta Med 2004; 70(2): 93–103.
14. Ammon HP: Boswellic acids in chronic inflammatory diseases. Planta Med 2006; 72(12): 1100–16.
15. Ahmad A, Khan A and Manzoor N: Antifungal potential of clove oil and its volatile vapors against Candida albicans. Phytomedicine 2011; 18(13): 1243–7.
16. Cowan MM: Plant products as antimicrobial agents. Clin Microbiol Rev 1999; 12(4): 564–82.
17. Cushnie TP and Lamb AJ: Recent advances in understanding the antibacterial properties of flavonoids. Int J Antimicrob Agents 2011; 38(2): 99–107.
18. Baruah S & Dey A: Morphological and anatomical characterization of Piper longum: A promising medicinal herb of Northeast India. In Compendium of Botanic Studies 2022; 1–10. ISBN 10: 9386302233.
19. Upadhya V & Kori VK: Pharmacognostical and phytochemical study of Piper longum and Piper retrofractum Vahl. Journal of Pharmacognosy and Phytochemistry 2016; 5(5): 1–6.
20. Sharma SV & Vinay R: Pharmacognostical and phytochemical study of Piper longum and Piper retrofractum Vahl. International Journal of Research in Ayurveda and Pharmacy 2012; 3(5): 1–6.
21. Chaudhary S & Shukla VJ: Pharmacognostical and pharmaceutical evaluation of Vasa Avaleha – An Ayurvedic compound. International Journal of Research in Ayurveda and Pharmacy 2015; 6(6): 1–6.
22. Mehra P & Puri HS: The pharmacognosy of Piper longum Indian Journal of Pharmaceutical Sciences 1977; 39(4): 161–165.
23. Chaudhary S & Shukla VJ: Pharmacognostical and pharmaceutical evaluation of Vasa Avaleha – An Ayurvedic compound. International Journal of Research in Ayurveda and Pharmacy 2015; 6(6): 1–6.
24. Mehra P & Puri HS: The pharmacognosy of Piper longum Indian Journal of Pharmaceutical Sciences 1977; 39(4): 161–165.
25. Chaudhary S & Shukla VJ: Pharmacognostical and pharmaceutical evaluation of Vasa Avaleha – An Ayurvedic compound. International Journal of Research in Ayurveda and Pharmacy 2015; 6(6): 1–6.
26. Suman F, Kumar D & Prakash O: Pharmacognostical, Physico-Chemical and Preliminary Phytochemical Analysis of Whole Plant of Piper longum International Journal of Research and Review 2021; 8(8): 440–449.
27. Singh S, Priyadarshi A, Singh B & Sharma P: Pharmacognostical and Phytochemical Analysis of Pippali (Piper longum). The Pharma Innovation Journal 2018; 7(6): 286–289.
28. Sharma SV & Vinay R: Pharmacognostical and Phytochemical Study of Piper longum and Piper retrofractum Vahl. Journal of Pharmacognosy and Phytochemistry CABI Digital Library 2012; 5(5): 1–6.
29. Dahanayake J & Jayasinghe L: Pharmacognostical, physico-chemical and phytochemical evaluation for standardization of three piper species used in ayurvedic medicine. Asian Journal of Pharmaceutical and Clinical Research 2019; 12(2): 1828–1832.
30. Ahadu Shareef, T H, Navabshan I, Divan Masood, M, Eswara Yuvaraj T, & Sherif A: Investigation of Phytochemicals, Spectral Properties, Anticancer, Antidiabetic, and Antimicrobial Activities of Chosen Ayurvedic Remedies 2024. arXiv. https://arxiv.org/abs/2412.17005arXiv
31. Benchamin D & Khandelwal KR: Pharmacognostical and Pharmaceutical Evaluation of Vasa Avaleha – An Ayurvedic Compound. International Journal of Research in Ayurveda and Pharmacy 2013; 6(6): 1–6.
32. Daniel M: A Critical Review on the Pharmacognosy and Chemistry of the Fruiting Spikes of Piper longum Indian Journal of Applied Research 2013; 3(2): 169–175.
33. Chaudhary S & Shukla VJ: Pharmacognostical and Pharmaceutical Evaluation of Vasa Avaleha – An Ayurvedic Compound. International Journal of Research in Ayurveda and Pharmacy 2015; 6(6): 1–6.
34. Mehra P & Puri HS: The Pharmacognosy of Piper longum Linn. Indian Journal of Pharmaceutical Sciences 1977; 39(4): 161–165.
35. Chaudhary S & Shukla VJ: Pharmacognostical and Pharmaceutical Evaluation of Vasa Avaleha – An Ayurvedic Compound. International Journal of Research in Ayurveda and Pharmacy 2015; 6(6): 1–6.
36. Uyangoda IS: Phytochemical screening, TLC fingerprinting, and GC-MS analysis of Piper longum and Piper sarmentosum Roxb. grown in Sri Lanka for validated herbal medicine applications: A comparative study. Scientific Research Network. Retrieved from 2025. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=5510705
37. Rathod SS & Singhal RS: Extraction of piperine from Piper longum using ultrasound. Ultrasonics Sonochemistry 2014; 21(1): 1–6. https://doi.org/10.1016/j.ultsonch.2013.04.003
38. Harborne JB: Phytochemical methods: A guide to modern techniques of plant analysis (3rd ed.). Springer Science & Business Media 1998.
39. Padma Priya NR & Stevens Jones RD: Larvicidal activity and GC–MS analysis of Piper longum leaf extract fraction against human vector mosquitoes (Diptera: Culicidae). International Journal of Mosquito Research, 2021; 8(4): 31–37. https://doi.org/10.22271/23487941.2021.v8.i4a.548
40. Rajopadhye AA, Namjoshi TP & Upadhye AS: Rapid validated HPTLC method for estimation of piperine and piperlongumine in root of Piper longum extract and its commercial formulation. Revista Brasileira de Farmacognosia 2012; 22(6): 1355–1361. https://doi.org/10.1590/S0102-695X2012005000113
41. Wickramaratne MN & Jayawardena N: In-vitro α-amylase inhibitory activity of the leaf extracts of Piper longum Pharmacognosy Journal 2016; 8(5): 427–431. https://doi.org/10.5530/pj.2016.5.9
42. Thapa CB, Bhattarai HD, Pant KK & Pant B: Comparative antioxidant, antibacterial, and antidiabetic activities of in-vitro grown callus and wild-grown various parts of Piper longum Phytomedicine Plus 2025; 4(2): 100586. https://doi.org/10.1016/j.phyplu.2024.100586
43. Sakat S, Juvekar AR & Gambhire MN: In-vitro antioxidant and anti-inflammatory activity of methanol extract of Oxalis corniculata International Journal of Pharmacy and Pharmaceutical Sciences 2010; 2(1): 146–155.
44. Akinmoladun FO, Olaleye TM & Farombi EO: Evaluation of in-vitro anti-inflammatory and antioxidant activities of selected plant extracts using HRBC membrane stabilization and other models. Journal of Ethnopharmacology 2021; 281: 114518. https://doi.org/10.1016/j.jep.2021.114518
45. Kumar R & Singh R: Evaluation of antifungal activity of Piper longum leaf extract against pathogenic fungi using agar well and disc diffusion methods. Asian Pacific Journal of Tropical Biomedicine 2017; 7(6): 567–571. https://doi.org/10.1016/j.apjtb.2017.05.003
46. Akinmoladun FO, Olaleye TM & Farombi EO: In-vitro antibacterial activity of Piper longum extracts against selected human pathogenic bacteria using agar well diffusion and microdilution methods. Journal of Ethnopharmacology 2020; 260: 112992. https://doi.org/10.1016/j.jep.2020.112992
    

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    How to cite this article:

    Abith SA, Prabha PM and Sangameswaran B: Pharmacognostical and phytopharmacological insights into Piper longum L. leaves with GC–MS and HPTLC fingerprinting. Int J Pharm Sci & Res 2026; 17(4): 1292-10. doi: 10.13040/IJPSR.0975-8232.17(4).1292-10.

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