Phytochemical investigation and biological activities of genus Corydalis

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dc.contributor.advisorDr. Ram Lal (Swagat) Shrestha
dc.contributor.authorMaharjan, Binita
dc.date.accessioned2025-01-05T05:06:59Z
dc.date.available2025-01-05T05:06:59Z
dc.date.issued2024-12
dc.description.abstractThe Himalayan region in Nepal is home to high-altitude flora known for their rich bioactive compounds. These compounds hold significant medicinal value and have been traditionally used to treat a variety of diseases. Among these, species of the Corydalis genus are recognized for their abundance of isoquinoline alkaloids, known for their diverse bioactivities. The three species, Corydalis chaerophylla, collected from Phulchowki, Lalitpur and C. govaniana, and C. casimiriana, collected from Langtang, Rasuwa, Nepal were extracted sequentially with hexane, methanol, and chloroform. The extracts of C. chaerophylla were subjected to liquid chromatography diode array detection multiple stage mass spectrometry (LC-DAD-MSn) analysis, resulting in the identification of fifteen alkaloids. Antimicrobial testing of extracts revealed significant inhibition with methanol and chloroform extracts, supported by high phenolic content (113 mg GAE/g in chloroform) and antioxidant activity (IC50 = 261.5 μg/mL in the DPPH assay). The methanol extract exhibited strong α-amylase inhibitory activity (IC50 = 51.52 μg/mL), highlighting its potential as an antidiabetic agent, but was less toxic in brine shrimp lethality analysis (LC50 = 196 μg/mL) compared to the chloroform extract. However, in vivo acute oral toxicity studies showed toxic effects for methanol (LD50 = 1000.36 mg/kg BW) and chloroform (LD50 = 515 mg/kg BW) extracts, requiring careful dose optimization for therapeutic applications. Chromatographic separation of chloroform extracts of different Corydalis species led to the isolation of thirteen pure alkaloids, including four new compounds from whose structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. The energy and force minimized molecular structures of these four compounds were obtained from quantum mechanical calculations using Becke, 3-parameter, Lee–Yang–Parr (B3LYP) functional and 6-31G* basis set at the density functional theory (DFT) level. The new identified compounds were named as Chaeronepaline-A (1), Chaeronepaline-B (2), Chaeronepaline-C (3), and Chaeronepaline-D (4), respectively, owing to the C. chaerophylla species from Nepal. Additionally, known alkaloids such as bicuculline (5), corydalmine (6), 8-hydroxydihydrosanguinarine (7), dihydrosanguinarine (8), and scoulerine (9) were isolated from C. chaerophylla, while govaniadine (10), was isolated and characterized from C. govaniana and stylopine (11), adlumine (12), and adlumidine (13) were identified from C. casimiriana. In a first set of experiment, berberine, californidine, and govaniadine demonstrated low-density lipoprotein receptor (LDLR) induction in human hepatocyte model similar to 2.5 μM simvastatin. Californidine and berberine decreased proprotein convertase subtilisin/kexin type 9 (PCSK9) expression, contrasting with simvastatin, which increased PCSK9. Govaniadine showed a statin-like effect by increasing both LDLR and PCSK9 levels. Further testing in the hepatocyte model showed that berberine, californidine, and govaniadine reduced total cholesterol levels, suggesting a promising hypocholesterolemic effect. Also, in the second set, the newly isolated alkaloids (1-4) were also evaluated in Huh7 cells to examine their impact on LDLR and PCSK9 expression and cholesterol biosynthesis for potential hypocholesterolemic activity. Results indicated that compounds 2 and 3 increased LDLR expression and inhibited cholesterol synthesis, with compound 2 additionally reducing PCSK9 secretion in Huh7 cells. Thus, this study identified and characterized bioactive alkaloids from Corydalis species, with compound 2 (chaeronepaline-B) demonstrating significant hypocholesterolemic potential by modulating LDLR, PCSK9, and cholesterol biosynthesis pathways. These findings highlight the therapeutic promise of Corydalis alkaloids, warranting further in vivo validation. नेपालको हिमाली क्षेत्रका उच्च भू-भागमा पाइने वनस्पतिहरू जैविक सक्रिय यौगिकहरूले भरिपूर्ण छन् । यी यौगिकहरूले महत्वपूर्ण औषधीय मूल्य राख्दछन् र परम्परागत रूपमा विभिन्न रोगहरूको उपचारका लागि प्रयोग हुँदै आएका छन् । Corydalis प्रजातिले जैविक सक्रियता देखाउनुका साथै यसमा आइसोक्विनोलिन एल्कालोइडहरू प्रचुर मात्रामा पाईन्छन् । नेपालबाट सङ्कलित Corydalis chaerophylla, C. govaniana र C. casimiriana को नमूनाहरूमा हेक्जेन, मिथानोल, र क्लोरोफर्म जस्ता घोलकको प्रयोग गरी सङ्कलित गरियो । C. chaerophylla का हेक्जेन, मिथानोल, र क्लोरोफर्मका एक्सट्राक्टहरूलाई LC-DAD-MSn विश्लेषण द्वारा १५ विभिन्न प्रकारका एल्कालोइडहरू पहिचान गरियो । मिथानोल र क्लोरोफर्म एक्सट्राक्टहरूले चार प्रकारका जीवाणु र एक ढुसी विरुद्ध महत्वपूर्ण गतिविधि देखाए, जसमा क्लोरोफर्म एक्सट्राक्टले उच्च फिनोलिक सामग्री (११३ mg GAE/g) र DPPH Assay मा उत्कृष्ट एन्टिअक्सिडेन्ट क्षमता (IC50 २६१.५ μg/mL) देखायो। मिथानोल एक्सट्राक्टले अल्फा–एमाइलेज अवरोध (IC50 ५१.५२ μg/mL) र कम विषाक्तता (LC50 १९६ μg/mL) देखायो, तर इन भिभो टक्सिसिटी परीक्षणले मिथानोल र क्लोरोफर्मका LD50 क्रमशः १०००.३६ र ५१५ mg/kg BW रहेको देखायो। तीनवटै बिरुवाहरूका क्लोरोफर्म एक्सट्राक्टहरूलाई क्रोमाटोग्राफिक विधिबाट १३ विभिन्न शुद्ध एल्कालोइडहरू अलग गरियो, जसमा Corydalis chaerophylla बाट चार नयाँ एल्कालोइडहरू पत्ता लगाइयो । यी नयाँ यौगिकहरूको संरचना 1D र 2D स्पेक्ट्रोस्कोपी र मास स्पेक्ट्रोमेट्री प्रयोग गरी व्याख्या गरियो । यी चार यौगिकहरूको ऊर्जा र बल न्यूनतम आणविक संरचनाहरू B3LYP कार्यात्मक र 6-31G* आधार DFT स्तरमा सेट प्रयोग गरेर क्वान्टम मेकानिकल गणनाहरूबाट प्राप्त गरियो । तिनीहरूलाई क्रमशः Chaeronepaline-A (१), Chaeronepaline-B (२), Chaeronepaline-C (३), Chaeronepaline-D (४) नाम दिइयो । यस्तै, C. chaerophylla बाट bicuculline (५), corydalmine (६), 8-hydroxydihydrosanguinarine (७), dihydrosanguinarine (८), scoulerine (९) जस्ता ज्ञात यौगिकहरू अलग गरिएको थियो, भने C. govaniana बाट govaniadine (१०) अलग गरी पहिचान गरिएको थियो र stylopine (११), adlumine (१२), तथा adlumidine (१३) लाई C. casimiriana बाट पहिचान गरिएको थियो । Berberine, californidine र govaniadine ले LDLR वृद्धि गर्ने गुण देखायो, जुन Simvastatine को तुलनामा लगभग समान थियो । Californidine र berberine ले PCSK9 को प्रभाव कम गरे, जबकि govaniadine ले स्टाटिन–जस्तै प्रभाव देखायो । नयाँ एल्कालोइडहरू (१–४) लाई Huh7 कोषहरूमा LDLR र PCSK9 को एक्सप्रेशन र कोलेस्ट्रोल बायोसिन्थेसिसमा तिनीहरूको प्रभाव मूल्याङ्कन गर्न परीक्षण गरियो । परिणामले यौगिक २ र ३ ले LDLR वृद्धि गरेको र कोलेस्ट्रोल संश्लेषण रोक्ने प्रतिक्रिया देखियो । यौगिक २ ले PCSK9 को स्राव पनि घटायो, जसले यसलाई सम्भावित हाइपोक्लोलेस्टेरोलिमिक एजेन्टको रूपमा संकेत गर्‍यो । यी निष्कर्षहरूले Corydalis एल्कलॉइडहरूको चिकित्सीय सम्भावनालाई प्रकाश पार्छ, जसले थप in vivo अनुसन्धानको आवश्यकता देखाउँछ।
dc.identifier.urihttps://hdl.handle.net/20.500.14540/23550
dc.language.isoen
dc.publisherInstitute of Sciece & Technology
dc.subjectCorydalis
dc.subjectExtraction
dc.subjectBioactivities
dc.subjectIsolation
dc.subjectCharacterization
dc.subjectIsoquinoline Alkaloids
dc.subjectAnticholesterol
dc.titlePhytochemical investigation and biological activities of genus Corydalis
dc.typeThesis
local.academic.levelPh.D.
local.institute.titleInstitute of Science & Technology
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