Botany

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Browsing Botany by Advisor "Prof. Dr. Bijaya Pant, Dr. Hari Datta Bhattarai, Dr. Krishna Kumar Pant"

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    Comparative Bioactivities of in Vivo and in Vitro Materials of Piper longum L. and Paris polyphylla Sm
    (Institute of Science & Technology, TU, 2024-11) Thapa, Chandra Bahadur; Prof. Dr. Bijaya Pant, Dr. Hari Datta Bhattarai, Dr. Krishna Kumar Pant
    Piper longum L. (Piperaceae) is a valuable tropical medicinal plant used to cure a variety of diseases. Similarly, Paris polyphylla Sm. (Melanthiaceae) is a temperate or subalpine vulnerable medicinal plant used to treat a variety of diseases. However, unsustainable utilization, illegal collection and export, habitat destruction, and climate change are threatening its survival in its natural habitats. As a result, their population in natural habitats is declining. On the other hand, most research activities focus on bioactivity and chemical analysis of in vivo parts; however, research on bioactivity and chemical analysis of in vitro-cultured calli is extremely limited. In the present study, the bioactivity of in vitro and in vivo cultured parts of P. longum and P. polyphylla were compared. The most effective medium for direct organogenesis by regeneration of multiple shoots (5.33±1.15) in P. longum from nodal explants was MS medium supplemented with 1.0 mg/L TDZ. The maximum number of roots (7.0±1.0) was regenerated in MS + 1.0 mg/L IBA from in vitro shoots. Plants were also regenerated by indirect organogenesis from leaf and node explants, and by somatic embryogenesis. Callus was induced from leaf explants in MS medium enriched with 2,4-D or 2.4-D + KN, while it was induced from nodal explants in MS media enriched with NAA only. Multiple shoots were obtained in MS + 0.25 mg/L TDZ (25.33±1.52) for leaf callus and 2.0 mg/L NAA (18.33±1.50) for nodal callus. Subsequently, the maximum number of roots was regenerated from in vitro shoots by using 0.5 mg/L IBA from the leaf callus and 2.0 mg/L NAA from the nodal callus. In addition, the maximum embryogenic/nodular calli with somatic embryos (SEs) were found in MS + 1.5 mg/L 2,4-D + 1.0 mg/L KN (28.33±3.05 SEs) for leaf callus, and 1.0 mg/L NAA (12.66±2.51 SEs) for nodal callus. Nodular calli with SEs were differentiated into plantlets/seedlings in MS + 0.5-2.5 mg/L TDZ and full-strength MS media without PGRs. Similarly, P. polyphylla was also propagated in vitro using leaf explants, in which the best media for callus formation and mini-rhizomes with root differentiation were MS + 0.25 mg/L 2,4-D + 0.5 mg/L KN and MS + 2.5 mg/L KN respectively. The dichloromethane (DCM) fraction of P. longum root showed the highest antioxidant activity (IC50: 134.81±1.16 µg/mL), the highest total phenolic content (TPC), and the highest total flavonoid content (TFC) compared to other extracts of in vivo parts and in vitro callus. It showed 100% growth inhibition against five bacterial strains, such as Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Bacillus subtilis, with the lowest minimum inhibitory concentration (MIC) of 5.0 mg/mL, and the minimum bactericidal concentration (MBC) of 8.35 mg/mL for S. aureus. It was more cytotoxic to brine shrimp nauplii (LC50: 156.78±5.49 µg/mL), while the crude extract of the fruit showed higher anticancer activity (IC50: 146.55±2.31 µg/mL) on U-2 OS and HeLa cell lines (LC50: 273.31±17.89 µg/mL). Similarly, the DCM fraction of the root showed higher inhibition of α-amylase (IC50: 365.21±31.021 µg/mL) and α-glucosidase (IC50: 489.07±27.966 µg/mL) compared to other extracts of in vivo parts and in vitro callus. In addition, callus suspension culture in MS medium showed that salicylic acid increased callus biomass compared to other treatments, while 100 mg/L phenylalanine resulted in the maximum antioxidant activity (IC50: 22.44±1.96 µg/mL), TPC, and TFC. Similarly, the DCM fraction of P. polyphylla showed the highest antioxidant activity (IC50: 197.06±0.635 µg/mL), TPC, and TFC compared to other extracts of rhizome and callus. It also showed 100% growth inhibition against the five bacterial strains tested, with the lowest MIC at 5.0 mg/mL and MBC at 7.5 mg/mL for E. coli. It was more cytotoxic on brine shrimp nauplii (LC50: 201.78±70.97 µg/mL). It also showed higher anticancer activity (IC50: 235.94±0.72 µg/mL) to HeLa cell lines, but the methanol fraction was more cytotoxic to MCF-7 cell lines (IC50: 211.36±0.57 µg/mL). However, the crude extract of the rhizome inhibited α-amylase (IC50: 95.45±8.71 µg/mL) and α-glucosidase (IC50: 51.40±8.62 µg/mL) more effectively than other extracts of rhizome and callus. In addition, suspension culture in MS medium revealed that salicylic acid increased callus biomass than other treatments, but liquid MS media supplemented with BAP+NAA resulted in the highest antioxidant activity, TPC, and TFC than phenylalanine or salicylic acid. LC-HRMS analysis of the DCM fraction of the root and crude extract of the callus of P. longum revealed a total of 27 compounds. Similarly, the DCM fraction of the rhizome and the crude extract of the callus of P. polyphylla exhibited a total of 31 compounds. Piper longum L. (पिपला) एक बहुमुल्य उष्णप्रदेशीय औषधीय गुण भएको बिरुवा हो, जुन विभिन्न प्रकारका रोगहरु निको पार्न प्रयोग गरिन्छ । त्यस्तै, Paris polyphylla Sm. (सतुवा) एक समशीतोष्ण वा हिमाली भागमा पाईने जोखिमयुक्त (Vulnerable) औषधीय गुण भएको बिरुवा हो, जुन विभिन्न प्रकारका रोगहरुको उपचार गर्न प्रयोग गरिन्छ । तथापि, दिगो उपयोगिताको अभाव, अवैध संकलन र निर्यात, बासस्थानको ह्रास र जलवायु परिवर्तनले प्राकृतिक बासस्थानमा यिनीहरुको अस्तित्वलाई खतरामा पारेको छ । फलस्वरुप, प्राकृतिक बासस्थानमा यिनीहरुको संख्या घट्दै गइरहेको छ । अर्कोतर्फ, अधिकांश अनुसन्धान गतिविधिहरु यी विरुवाहरुका प्राकृतिक परिवेशमा (in vivo) उत्पादन भएका विभिन्न भागहरुका bioactivity र रसायनिक विश्लेषणमा केन्द्रित छन् । तर तन्तु प्रविधिबाट (tissue culture) उत्पादित callus को bioactivity र रसायनिक विश्लेषणको अनुसन्धान अत्यन्त सीमित छ । तसर्थ, यस अनुसन्धानमा पिपला र सतुवाको प्राकृतिक परिवेशमा (in vivo) उत्पादन भएका विभिन्न भागहरु र कृत्रिम परिवेशमा (in vitro) उत्पादित callus को तुलनात्मकरुपमा bioactivity र रसायनिक विश्लेषणको अध्ययन गरिएको छ। Tissue Culture प्रविधि प्रयोग गरि कृत्रिम पोषक माध्यम (MS media) मा पिपलाको काण्डको आँख्ला (node) र पात (leaf) बाट स-साना बिरुवाहरु उत्पादन गरियो, अनि सतुवाको पातबाट पनि यसका बिरुवाहरु उत्पादन गरियो । पिपलाको आँख्लाबाट बिरुवाहरु उत्पादन गर्दा प्रत्यक्ष organogenesis विधिवाट र यसको पातबाट र सतुवाको पातबाट बिरुवाहरु उत्पादन गर्दा अप्रत्यक्ष organogenesis र somatic embryogenesis उपयुक्त देखियो । त्यसैगरी, पिपलाको आँख्ला र callus बाट धेरै संख्यामा टुसाहरु (Shoots) उत्पादन गर्नको लागी र सतुवाको callus बाट धेरै संख्यामा टुसाहरु (Mini-rhizome) उत्पादन गर्नको लागी MS पोषक माध्यममा TDZ र KN हर्मोनहरु उपयुक्त देखियो । साथै टुसाबाट जराहरु उतपादन गर्नको लागी MS पोषक माध्यममा IBA हर्मोन उपयुक्त देखियो । पिपलाको जराको Dichloromethane (DCM) extract ले अरु extract ले भन्दा बढी antioxidant activity, total phenolic content (TPC) र total flavonoid content (TFC) देखायो । यस extract ले ५ प्रकारका ब्याक्टेरियाहरु (Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus र Bacillus subtilis) को बृद्धिलाई रोकेको देखायो । त्यसैगरी, यसले Brine shrimp झिंगाका लार्भाहरुलाई सबैभन्दा कम मात्राको प्रयोगमा मारेको पाईयो । तर पिपलाको फलको crude extract ले अन्य extract ले भन्दा हड्डि र पाठेघर क्यान्सर कोषहरु प्रति उच्च क्यान्सर प्रतिरोध देखायो । जराको DCM extract ले मधुमेह रोगसंग सम्बन्धित α-amylase र α-glucosidase ईन्जाईम प्रति उच्च प्रतिरोध प्रदर्शन गर्यो । यसबाहेक, पिपलाको callus लाई MS तरल पोषक माध्यममा salicylic acid प्रयोग गरी culture गर्दा यसको तौल (biomass) अरुको तुलनामा बढाएको पाईयो, जबकि 100 mg/L phenylalanine तरल माध्यममा प्रयोग गर्दा सबैभन्दा बढी antioxidant activity, TPC, र TFC को मात्रा पाईयो । तसर्थ, tissue culture माध्यमबाट उत्पादित callus ले bioactive compound हरु उत्पादन गर्यो, जसले गर्दा anti-oxidant, antibacterial, anticancer र antidiabetic प्रतिक्रिया देखायो । त्यसैगरी, सतुवाको राईजोमको DCM fraction ले सबैभन्दा बढी antioxidant activity, TPC र TFC को मात्रा देखायो । यसले परिक्षण गरिएका सबै ५ प्रकारका ब्याक्टेरियाहरुको १००% बृद्धि प्रति अवरोध देखाएको छ । यसले Brine shrimp झिंगाका लार्भाहरुलाई सबैभन्दा कम मात्राको प्रयोगमा मारेको पाईयो । साथै यस extract ले अन्य extract ले भन्दा छातीका क्यान्सर कोषहरु प्रति उच्च प्रतिरोध देखायो । त्यसैगरी, राईजोमको crude extract ले मधुमेह रोगसंग सम्बन्धित α-amylase र α-glucosidase ईन्जाईम प्रति उच्च प्रतिरोध प्रदर्शन गर्यो । यसबाहेक, सतुवाको callus लाई MS तरल पोषक माध्यममा salicylic acid प्रयोग गरी culture गर्दा यसको तौल (biomass) अरुको तुलनामा बढेको पाईयो, जबकि MS media मा NAA र BAP हर्मोन प्रयोग गरी तरल माध्यममा culture गर्दा सबैभन्दा बढी antioxidant activity, TPC, र TFC को मात्रा पाईयो । तसर्थ, salicyclic acid र phenylalanine लाई callus suspension culture मा प्रयोग गर्दा secondary metabolite बढेको पाईयो । LC-HRMS प्रविधी प्रयोग गरी पिपलाको DCM fraction र callus को crude extract को रसायनिक परिक्षण गर्दा कुल २७ वटा रसायनिक यौगिकहरु पाईयो, तर सतुवाको राईजोमको DCM fraction र callus को crude extract को रसायनिक परिक्षण गर्दा कुल ३१ वटा रसायनिक यौगिकहरु पाईयो । केहि रसायनिक यौगिकहरु दुवै callus र विरुवाका in vivo भागहरु (जस्तै: जरा, राइजोम) मा पाइएको हुनाले यस अध्यनमा callus ले रसायनिक यौगिकहरु उत्पादन गरेको पाइयो ।