1 Influence of solvent polarity on phytochemicals, antioxidants and antimicrobial properties

2 of Delphinium denudatum: a medicinal herb from Sainj Valley, Himachal Pradesh, India
3
4 Abstract:
5
6 Plants with medicinal properties play an important role in pharmaceutical industries for their
7 disease prevention and treatment functions. Delphinium denudatum, commonly known as
8 jadwar, is an important medicinal plant of the Himalayan region. Therefore, in the present study,
9 the effect of solvent polarity (using seven different solvents i.e., methanol, ethanol, acetone,
10 chloroform, ethyl acetate, hexane, and water separately) was estimated for its secondary
11 metabolites production, antioxidants, and antimicrobial activity. Among the seven different
12 extracting solvents used, the methanol extract of leaf rendered the highest phenolic content
13 (80.52 mg GAE/g (dry weight (dw)). Acetone extracts for the shoot were found to be most
14 efficient with the extraction of the highest flavonoid content (57.53 mg QE/g (dw) while the
15 methanol extract of root rendered the highest tannin content 18.78 mg TAE/g (dw). Likewise, the
16 methanol extract of the leaf showed the highest flavonol content 34.76 mg QE/g (dw). For
17 antioxidant activity, the IC50 value for ABTS activity ranged from 35.15 to 103.08 µg/mL and for
18 DPPH activity it was 75.23 to 256.21 µg/mL. Further, all the plant parts i.e., leaf, shoot, and root,
19 showed antimicrobial activity against Bacillus subtilis, Escherichia coli, and Serratia
20 marcescens having MIC between 400 to 900 µg/mL. Among all the tested plant parts, polar
21 solvent leaf extracts had higher antioxidant activity. Furthermore, leaf, shoot, and root phenols,
22 flavonols, tannins, and ABTS activity have shown a positive relation with solvent polarity. In all
23 three plant parts phenols, flavonols, and tannin are positively correlated with antibacterial
24 activity. The present study further revealed that the secondary metabolites in the leaf, shoot, and
25 root extracts of Delphinium denudatum are an excellent source of antioxidant and anti-microbial
26 activity, thus validating the species’ therapeutic potential.

27 Keywords: Delphinium denudatum, medicinal herb, traditional medicine, antioxidants,

28 antimicrobials

29
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34
35
36 1. INTRODUCTION
37
38 Since natural products are more affordable and have fewer negative effects, their use is
39 becoming more and more popular. Meanwhile, plant extracts are said to be a source of several
40 phytochemicals, natural antioxidants, and antimicrobials, they are being investigated for
41 potential novel drugs [1]. Moreover, plants with medicinal properties play an increasingly
42 important role in pharmaceutical industries for their functions in disease prevention and
43 treatment by having potential antimicrobial activity [2]. A large group of compounds produced
44 by plants referred to as phytochemicals possessing high antioxidant properties have been seen to
45 help tackle numerous diseases. Scientific evidence suggests that antioxidants reduce the risk for
46 chronic diseases including heart and cancer diseases [3].
47
48 Phytochemical antioxidants are very efficient scavengers of free radicals. Antioxidants are
49 those substances that possess free radicals chain reaction-breaking properties. Plant products can
50 be attributed to the biological activities of their phytochemicals and antioxidant constituents such
51 as phenolic compounds, proanthocyanidin, vitamins, carotenoids, flavonoids, and saponin. [4].
52 Medicinal plants, especially the endemic and edible plants of a region, due to their ability to
53 produce natural compounds with antioxidant capacity and antimicrobial properties and due to
54 their health benefits are particularly important for the development of new drugs. In this
55 perspective, plant-based antimicrobials (derived from medicinal plants, in particular) are
56 increasingly receiving attention for harnessing their potential in the production of antimicrobial
57 substances, as safer sources of antibiotics. Crude extracts and essential oils of medicinal plants
58 possess bioactive compounds, often with antimicrobial and antioxidant properties [5].
59 Antimicrobial compounds are used in various areas such as pharmaceuticals, neutraceuticals,
60 textiles, dairy products, cosmetics, and personal care products [6].
61
62 Delphinium denudatum Wall (family Ranunculaceae) is an important medicinal plant
63 commonly known as jadwar. It provides one of the important drugs used as indigenous medicine

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64 in India, especially in Unani medicines [7; 8]. It is a perennial branched erect herb found in
65 Northwest Himalayas. Roots of D. denudatum are used for the treatment of toothache,
66 rheumatism, syphilis, snakebite, and aconite poisoning. It is also used as an alternative, tonic,
67 and in the treatment of epilepsy in the Unani healthcare system [9]. Images of D. denudatum
68 from the wild and in the laboratory are shown in Figure 1A. Ethanol and aqueous extracts are
69 reported to have morphine addiction activities [10; 11]. The ethanolic extract is also reported to
70 have a protective effect in rat models of Parkinson's disease [12]. The roots are reported to
71 contain diterpene alkaloids of cuisine and veatchite type [13]. Furthermore, before the extraction
72 of antioxidant and antibacterial compound(s), the influence of solvent polarity and optimization
73 of plant extractive values is particularly critical. Researchers have also focused on choosing
74 appropriate extraction solvents and extraction techniques for evaluating bioactive substances,
75 such as antimicrobials, from various medicinal plants and plant parts [6]. Despite the high
76 demand and widespread use of D. denudatum, there aren't many papers on the plant. For this
77 reason, the current work focuses on a comprehensive analysis of how solvent polarity impacts
78 phytochemical, antioxidant, and antibacterial activity. We have concentrated on solvent polarity
79 from polar to non-polar in this work because it is an important first step in the extraction process
80 of secondary metabolites. Its basic phytochemical and antibacterial activities make it an
81 important medicinal plant. This is important since the species grows throughout a wide range of
82 altitude and habitat types. Given the current context, the aim of the study is to examine how
83 different solvent systems affect the phytochemicals, antioxidants, and antibacterial qualities of
84 D. denudatum plant parts.
85
86 2. MATERIAL AND METHODS
87
88 2.1. Study site and Sample collection
89 The fresh and healthy root, stem, and leaves of D. denudatum were collected from Sainj Valley
90 of Kullu District in Himachal Pradesh (31⁰45.881' N to 31⁰76.897' N latitudes and 77⁰19.031' E
91 to 77⁰33.747' E longitudes) during May and June month of 2021; samples were brought to the
92 laboratory, air dried, converted into fine powder, and stored at 4-8 °C for the further analysis.
93
94 2.2. Extraction

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 95 Variation in extraction methods usually depends on the length of the extraction period, the
96 solvent used, and the solvent-to-sample ratio. The basic principle is to grind the plant material
97 finer, which increases the surface area for extraction thereby increasing the rate of extraction. In
98 this study, the extraction of plant material was achieved by homogenizing the plant tissue in
99 various solvents. Root, leaf, and stem were extracted (separately) in seven solvents (methanol,
100 ethanol, acetone, chloroform, ethyl acetate, hexane, and water separately) taking in a ratio of 1:5
101 (dry powder: solvent). The mouth of the conical flask was sealed with para-film. Samples were
102 macerated in a rotary shaker (Remi) at 160 rpm for 48 h.
103
104 2.3. Phytochemical properties of D. denudatum extracts
105 2.3.1. Total phenols
106 The total phenolic content of the extract was determined by the Folin-Ciocalteu method [14].
107 Briefly, 200 μL of crude extract (1 mg/mL) was made up to 3 mL with distilled water, mixed
108 thoroughly with 0.5 mL of Folin-Ciocalteu reagent for 3 min, followed by the addition of 2 mL
109 of 20% (w/v) sodium carbonate. The mixture was allowed to stand for a further 60 min in the
110 dark, and absorbance was measured at 650 nm. The total phenolic content was calculated from
111 the calibration curve, and the results were expressed as mg of gallic acid equivalent per gram of
112 dry weight (mg/g (dw)).
113
114 2.3.2. Total tannins
115 The tannin content of the extract was determined by the Folin-Denis method [15]. 50 µL of
116 crude extract was mixed properly with 0.5 mL of Folin Denis reagent, followed by the addition
117 of 7% sodium carbonate. 3 mL of distilled water was added to it and then the mixture was
118 allowed to stand for a further 20 min and absorbance was measured at 700 nm. The tannin
119 content was calculated from the calibration curve, and the results were expressed as mg of tannic
120 acid equivalent per g dry weight.
121
122 2.3.3. Total flavonoids
123 The aluminum chloride colorimetric method was used for the determination of the total
124 flavonoid content of the sample [16]. 0.5 ml of the extract (5 g/L) was mixed with 1.5 mL of
125 methanol and then, 0.1 mL of 10 % aluminum chloride was added, followed by 0.1 mL of
126 potassium acetate and 2.8 ml of distilled water. The mixture was incubated at room temperature

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127 for 30 min. The absorbance was measured by a spectrophotometer at 415 nm. The results were
128 expressed as milligrams of Quercetin equivalents (QE) per gram of extract (mg QE/g dry
129 extract). Quercetin was used as positive control and the standard curve was prepared by
130 quercetin in different concentrations.
131
132 2.3.4. Total flavonols
133 The flavonol content of the samples was determined since flavonols represent the main fraction
134 of compounds responsible for the antioxidant activity. The content was estimated according to
135 Tapan [17]. 2.0 mL of the sample, dissolved in ethanol, was mixed with 2.0 mL AlCl 3 (2%)
136 prepared in ethanol (96%) and 3.0 mL sodium acetate solution (50 g/L). The absorbance at 440
137 nm was recorded after 2.5 h incubation at 20 °C. The results were expressed as milligrams of
138 Quercetin equivalents (QE) per gram of extract (mg QE/g dry extract). Quercetin was used as
139 positive control and the standard curve was prepared by quercetin in different concentrations.
140
141 2.4. Antioxidant activity
142 2.4.1. DPPH assay
143 The free radicals-scavenging potential of crude extracts was determined using a DPPH assay
144 [18]. A 0.50 uL aliquot of the extract solution was mixed thoroughly with 2.5 mL of 0.3 mM
145 DPPH prepared in methanol for 1 min and then allowed to stand at room temperature for 20 min.
146 Absorbance was measured at 517 nm. Quantifying radical activity was determined based on a
147 standard curve of ascorbic acid prepared in methanol.
148
149 2.4.2. ABTS assay
150 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay was
151 carried out according to the method of [19]. ABTS·+ cation radical was produced by the reaction
152 between 7 mM ABTS in water and 2.45 mM potassium persulphate (1:1), stored in the dark at
153 room temperature for 12-16 h before use. ABTS·+ solution was then diluted with methanol to
154 obtain an absorbance of 0.700 at 734 nm. After the addition of 5 μL of plant extract to 3.995 mL
155 of diluted ABTS·+ solution; the absorbance was measured at 30 min after the initial mixing. An
156 appropriate solvent blank was run in each assay.
157
158 2.5. Antimicrobial activity

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159 2.5.1. Qualitative test (Plate based bioassay)
160 The bacterial cultures, Gram-positive i.e., Bacillus subtilis, and Gram-negative namely
161 Escherichia coli and Serratia marcescence were used as test organisms in this investigation. The
162 media used for the antibacterial test was Tryptone yeast extract Agar. The antibacterial activity
163 was carried out by employing 24h of cultures [6]. The activity of all the extracts was screened
164 for antibacterial activity and was tested separately using the disk diffusion method. About 30mL
165 of the agar medium with respective strains of bacteria was transferred aseptically into each
166 sterilized Petri plate. The extracts were placed in a 6 mm diameter. Antibacterial assay plates
167 were incubated at 37°C for 24-48 h and the diameter of the zone of inhibition was measured.
168
169 2.5.2. Quantitative test (Minimum Inhibitory Concentration)
170 Bacterial culture suspensions were prepared in TYE broth. For determination of MIC, 500 µL
171 standard compound solution of different concentrations ranging from 10 to 50 μg/mL was
172 diluted using 500 µL test organism and 4 mL TYE broth in the sterile test tube and then
173 incubated at 27 °C for 24-48 h. Control was prepared in two sets, one containing broth medium
174 and test organism while the other containing broth medium and extract [6]. After 24h, the MIC
175 values were recorded based on the lowest concentration showing an absence of growth in the
176 tubes. The test was further confirmed by plating on TYE agar.
177
178 2.6. Statistical analysis
179 Estimating phytochemical compounds (total phenolics, tannins, flavonols, and flavonoids),
180 antimicrobial and antioxidant activities by 2, 2- azinobis 3-ethylbenzothiazo line-6-sulphonic
181 acid, 1,1-diphenyl- 2 picrylhydrazyl were conducted in triplicates. The data expressed as the
182 means ± standard errors (SE) from experiments performed in triplicate. Statistical significance
183 and mean between groups were tested using Student’s t-test and two-way ANOVA. The p-value
184 <0.05 was considered significant. Pearsons’s correlation was calculated among different
185 parameters used in the present study using R software (R-3.4.1).
186
187 3. RESULTS AND DISCUSSION
188
189 3.1. Extraction yield

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190 The extract yield varied with different solvents, and the highest extract yield was obtained in
191 polar protic solvents such as methanol (45.21± 0.75% in shoot; 42.35± 0.84% in root and; 40.23
192 ± 0.89% in leaf) and ethanol (46.25± 0.86% in shoot; 36.12± 0.71% in root and; 35.26± 0.73% in
193 leaf) than polar aprotic solvents such as acetone (16.48± 0.26 % in root; 15.32± 0.29% in leaf
194 and; 13.15± 0.32% in leaf) and ethyl acetate (42.39± 0.69% in roots; 20.45± 0.69% in shoot and
195 19.23± 0.49% in leaf). The extraction yield showed a clear linkage between the polarity of the
196 solvent and yield. The polar protic solvent (methanol and ethanol) extracted a significantly
197 greater yield than the polar aprotic (acetone, aqueous, and ethyl acetate) and non-polar
198 (chloroform and hexane) solvents. The highest yield in methanol was due to the dissolving
199 efficacy of the organic compound with a protonatable functional group and low-molecular-
200 weight organic compound groups [6; 20]. Results on extract yield are shown in Figure 1B.
201
202 3.2. Phytochemical analysis
203 Phytochemical analysis showed the presence and absence of certain chemical constituents in
204 different extracts such as methanol, ethanol, ethyl acetate, acetone, hexane, chloroform, and
205 water. The total phenolic, flavonoids, flavonol, and tannin content of sample extracts obtained
206 from different plant parts of Delphinium denudatum varied with the solvent used for the
207 extraction (Table 1).
208
209 The concentration of total phenolic in the examined extract ranged from 10.45 to
210 80.52mg GAE/g/ dw. Among the seven different extracting solvents used, the methanol extract
211 of the leaf rendered the highest phenolic content of 80.52 mg GAE/g/dw followed by the ethanol
212 extract of leaf at 78.73 mg/GAE/g/dw, ethanol extract of shoot 74.84 mg GAE/g/dw, ethyl
213 acetate of root 67.45mg GAE/g/dw and aqueous extract & hexane showed lowest phenolic
214 contents than those of other solvents (Figure 2).
215
216 Flavonoids, tannin, and phenolic substances are constituents of plants with potential
217 antioxidant activity, mainly because they act as free radical scavengers [21]. The total flavonoid
218 content values examined ranged from 8.99 to 57.53 mg QE/g/dw. Amongst all the solvents tried
219 acetone extracts for the shoot was found to be most efficient with the extraction of the highest
220 flavonoid content (57.53 mg QE/g/dw), followed by chloroform of leaf (42.40 mg/QE/g/dw) and

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221 chloroform of shoot (42.14 mg/QE/g/dw) and ethyl acetate extract showed lowest flavonoid
222 content than those of other solvents (Table 1 and Figure 2).
223
224 Plants are rich sources of active metabolites like tannins, alkaloids, flavonoids, phenols,
225 steroids, etc. which are responsible for their therapeutic activities [4]. Plants are a potent source
226 of phytochemical constituents that are responsible for their pharmacological activities. Many
227 phytochemical compounds, such as phenolic, flavonoids, tannin, and alkaloids have been
228 isolated from various plant species [1-3].
229
230 The concentration of tannin in the examined extract ranged from 1.37 to 18.78 mg
231 TAE/g/ dw. Among the seven different extracting solvents used the methanol extract of root
232 rendered the highest tannin content of 18.78 mg TAE/g/dw followed by ethyl acetate extract of
233 leaf at 17.79 mg/TAE/g/dw, ethanol extract of root 15.87 mg TAE/g/dw, ethyl acetate of root
234 15.13 mg TAE/g/dw and aqueous extract showed lowest tannin contents than those of other
235 solvents (Figure 2).
236
237 Figure 2 showed that the total flavonol concentration of different extracts ranged from
238 10.83 to 34.76 mg QE/g/dw. The methanol extract of the leaf showed the highest flavonol
239 content 34.76 mg QE/g/dw, followed by the methanol extract of the shoot (30.40 mg QE/g/dw),
240 and ethyl acetate extract showed the lowest flavonol contents as compared to other solvents.
241 According to Mohanpriya and Siva [22], the phytochemical screening of different solvent
242 extracts (like flavonoids, tannin, phenolic, etc.) of the root of D. denudatum revealed the
243 presence of medically bioactive constituents and tannin is absent in the D. denudatum ethanolic
244 extract. However, the present study showed that tannin is present in the ethanolic extract of D.
245 denudatum.
246
247 Medicinal plants can be used in fresh or dried form. However, drying is the most
248 common method for the post-harvest preservation of medicinal plants and must be accomplished
249 as soon as possible after harvesting to increase the quality of plants and to prevent the expected
250 contamination and losses [23]. Previously Delphinium malabaricum showed the presence of
251 high phenolics and alkaloids content in root extracts whereas the leaf extract exhibited the
252 presence of high flavonoids content [24], but in the present study, D. denudatum showed that the

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253 presence of high phenolic content in leaf extracts. Interestingly, this is the first report on the
254 presence of total phenolics, flavonoids, tannins, and flavonols in the various solvent extracts of
255 the leaf, shoot, and root of D. denudatum which have not yet been documented by many
256 researchers.
257
258 3.3. Antioxidant activity
259 DPPH is a stable free radical at room temperature and accepts an electron, and hydrogen radical
260 to become stable diamagnetic molecules [25]. The reduction capability of the DPPH radical is
261 determined by the decrease in its absorbance at 517 nm, induced by antioxidants. In the present
262 study, Delphinium plant extracts' shoot and leaf were examined for their radical scavenging
263 properties of DPPH and ABTS. Results showed that among all the tested solvent extracts,
264 methanol, ethanol, ethyl acetate, and acetone showed antioxidant potential. For antioxidant
265 activity, the methanol extract of the shoot showed the highest antioxidant value at 43.44±0.96
266 mg AAE/g (dw) followed by the ethanolic extract of root at 43.00±0.53 mg QE/g (dw), and
267 ethanolic extract of leaf 35±0.44 mg QE/g (dw). Likewise for DPPH scavenging activity, the
268 highest activity present in ethyl acetate extract of shoot was 15.43±0.032 mg AAE/g (dw) and
269 root 15.25±0.068 mg AAE/g (dw), methanol extract of leaf 12.48±0.0105 mg AAE/g (dw)
270 (Figure 3). IC50 value for ABTS activity is ranged from 35.15 to 103.08 µg/mL and for DPPH
271 activity it is 75.23 to 256.21 µg/mL (Table 2). Comparable outcomes were noted for several
272 therapeutic plants, including Celastrus peniculatus Larrea tridentate, Peltophorum ferrugineum,
273 and Ocimum gratissimum [26; 27].
274
275 ABTS is an excellent substrate for peroxidase and is frequently used to study the antioxidant
276 properties of natural compounds [28]. Antioxidants are essential substances that possess the
277 ability to protect the body from damage caused by free radical-induced oxidative stress.
278 Medicinal plants are a rich source of phytochemicals, such as carotenoids, flavonoids, and other
279 phenolic compounds having high free-radical scavenging activity, which helps to reduce the risk
280 of chronic diseases, such as cardiovascular disease, cancers, and neuronal degeneration [29].
281
282 3.4. Antimicrobial activity
283 All three plant parts (root, shoot, and leaf) were used for testing the antibacterial activity against
284 both Gram-positive i.e., Bacillus subtilis, and Gram-negative bacteria i.e., Escherichia coli and

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285 Serratia marcescens. Ethyl acetate, chloroform, and aqueous extracts showed selective
286 antibacterial activity. Results in detail are given in our previous study [30]. Moreover,
287 methanolic, ethanolic, and chloroform extracts showed antimicrobial activity against all the
288 tested bacteria. Only chloroform extracts of shoot didn’t show activity against Gram-negative
289 bacteria i.e., Escherichia coli and Serratia marcescens. Results on the Zone of inhibition (mm)
290 are shown in Table 3. Methanol and ethanol extracts are reported for good antibacterial potential
291 in previous reports also from Taxus wallichiana [6] and Curcuma caesia [3]. Quantitative
292 analysis was also done by calculating the minimum inhibitory concentration of all the plant
293 extracts having the zone of inhibition. MIC ranged between 500 to 900 µg/mL for Bacillus
294 subtilis, 500 to 600µg/mL for Escherichia coli, and 400 to 900 µg/mL for Serratia marcescens.
295 MIC in the same range was also reported from leaf, rhizome and in vitro propagated callus of
296 Paeonia emodi [1].
297
298 3.5. Pearsons Correlation
299 A Pearson's correlation analysis was conducted between various metrics, including solvent
300 polarity, phytochemicals, antioxidant activity, and antibacterial activity of individual plant
301 components. The outcomes are displayed in Figure 4. The phenol concentration and ABTS
302 activity of leaf solvent were positively linked with polarity. Phenolic and flavonoid content
303 positively correlated with antimicrobial activity. Further, solvent polarity, phenol concentration,
304 ABTS, and DPPH activity were all positively connected with solvent polarity. Phenolic and
305 flavonoid content positively correlated with antimicrobial activity. Likewise, phenol, tannin
306 concentration, ABTS activity, and antibacterial activity were all positively connected with
307 solvent polarity in roots. Phenolic and tannin concentrations positively correlated with
308 antimicrobial activity.
309
310 It has been found that phenol and flavonoids possessed antibacterial properties against
311 both gram-positive and gram-negative bacteria. Phenolic compounds' primary mechanisms of
312 action include damaging bacterial cell membranes, preventing the development of virulence
313 traits, and preventing the formation of bacterial biofilm [31]. Different processes are used by
314 flavonoid compounds to eliminate microorganisms. The presence of the phenolic hydroxyl
315 group in flavonoids has been shown to have a key role in inhibiting the formation of microbial
316 enzymes and simultaneously destroying the cell wall leading to cell death. This is achieved by

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317 increasing the protein binding affinity of flavonoids [32]. By promoting the breakdown of the
318 plasma membrane and impeding the growth of cell walls and protein synthesis, flavanols
319 eradicate fungi [33].
320
321 4. CONCLUSION
322 This is the first report to assess how different solvents affect the antioxidants, antimicrobial
323 activity, and phytochemical compositions of leaf, shoot, and root extracts of D. denudatum. The
324 results of this work indicate that more secondary metabolites with potential for antioxidant and
325 antibacterial activity can be produced by synthesizing secondary metabolites in Delphinium
326 denudatum leaf, shoot, and root extracts in polar solvents. The plant extracts of the leaves,
327 shoots, and roots of D. denudatum are rich in antioxidants and antibacterial compounds that can
328 be applied to pharmaceuticals, healthcare goods, medicinal applications, and nutritional needs
329 (such as food additives and preservatives). Future research should be done to find out whether
330 they have anti-diabetic, anti-proliferative, and anti-cancerous properties. Antioxidants and
331 antimicrobials can be extracted from the leaves and added to culinary and medical items. This
332 will support the preservation of it as a non-destructive harvesting technique.
333
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470

471 Figure 1: A) Images of Delphinium denudatum in wild and plant image (including all plant parts)
472 in lab. B) Extractive yield of D. denudatum different plant parts (Leaf, Shoot and Root).

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473 Figure 2: Phytochemical analysis of D. denudatum different plants parts extracts.

474 Figure 3. Effect of solvent polarity in antioxidants potential of D. denudatum different plants
475 parts extracts.

476 Figure 4. Pearsons Correlation among solvent polarity, phytochemical, antioxidants and
477 antimicrobial activity of different plants parts of Delphinium denudatum. Correlation is
478 significant at 0.05 level, grey highlighted box=p<0.05, simple box=p>0.05. ABTS= 2,2′-azino-
479 bis (3-ethylbenzothiazoline-6-sulfonic acid), DPPH= 2,2-Diphenyl-1-picrylhydrazyl, BS=
480 Bacillus subtilis, SM= Serratia marcescens and EC=Escherichia coli.

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481

482 Figure 1.

483

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484

485 Figure 2.

486

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487

488 Figure 3.

489

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490

491

492 Figure 4.

493

494

495

496

497

498

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499 Table 1. Qualitative phytochemical analysis of Delphinium denudatum different plants parts extracts.

Plant parts Solvent Phenolics Flavonoids Terpenoids Alkaloids Glycosides Saponins Protein Carbohydrates
Leaf Methanol ++++ ++++ ++++ ++++ ++++ ++++ ++ ++
Ethanol ++ ++ ++ ++ ++ - ++ ++
Ethyl acetate ++ ++ +++ ++ ++ +++ ++ ++
Acetone +++ ++ - + + - ++ ++
Chloroform + + ++ - + ++ - -
Hexane + + ++ - + - ++ ++
Aqueous ++ ++ ++ + + +++ + +
Shoot Methanol ++++ ++++ ++++ ++++ ++++ ++++ ++ ++
Ethanol + + ++ + ++ - ++ ++
Ethyl acetate ++ ++ +++ ++ ++ +++ ++ ++
Acetone +++ ++ - - + - ++ ++
Chloroform + + ++ - + ++ - -
Hexane + + ++ - + - ++ ++
Aqueous ++ ++ ++ +++ + +++ + +
Root Methanol ++++ ++++ ++++ ++++ ++++ ++++ ++ ++
Ethanol + ++ +++ - ++ - ++ ++
Ethyl acetate ++ ++ +++ ++ ++ +++ ++ ++
Acetone +++ ++ - - + - ++ ++
Chloroform + + ++ - + ++ - -
Hexane + + ++ - + ++ ++ ++
Aqueous ++ ++ ++ ++ + +++ + +
-= negative, += positive, number of + denotes colour intensity. + += light; + + +=medium; and + + + +=dark colour production.
500

501

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502 Table 2. Antioxidant activity of Delphinium denudatum different plants parts extracts.

Solvent Plant Parts IC50 (µg/mL) IC50 (µg/mL)

ABTS DPPH
Methanol Leaf 50.63±0.55 70.23±0.95
Shoot 35.15±0.95 95.19±0.75
Root 35.26±1.09 105.32±1.56
Ethanol Leaf 35.26±0.75 75.23±0.92
Shoot 60.24±0.72 90.10±0.79
Root 49.32±1.18 106.32±1.06
Ethyl acetate Leaf 95.32±0.65 95.32±0.29
Shoot 63.12±0.98 165.23±0.75
Root 54.23±1.06 115.23±1.32
Acetone Leaf 86.08±2.15 135.26±0.67
Shoot 103.08±1.26 156.32±0.49
Root 125.25±1.42 256.21±2.35
503

504

505

506

507

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508 Table 3. Antimicrobial Activity Delphinium denudatum different plants parts extracts.

509

Solvent Plant Parts Bacillus subtilis Escherichia coli Serratia marcescens

Zone of inhibition MIC (µg/mL) Zone of inhibition MIC (µg/mL) Zone of inhibition MIC (µg/mL)
Methanol Root 3.00±0.12 500 3.33±0.04 500 4.67±0.11 400
Shoot 2.33±0.11 700 1.67±0.10 500 2.61±0.08 900
Leaf 1.67± 0.04 800 6.67 ± 0.08 500 8.00±0.51 500
Ethanol Root 2.33±0.1 700 4.13±0.25 600 9.33±0.18 300
Shoot 7.00 ± 0.35 400 5.33±0.08 500 15.33± 0.11 300
Leaf 3.00± 0.07 600 6.67 ± 0.08 500 6.33 ± 0.36 300
Chloroform Root 2.33±0.11 900 4.67±0.15 700 2.0±0.07 900
Shoot 2.00±0.07 900 ND ND ND 900
Leaf 2.00±0.07 900 4.33±0.15 600 3.67±0.18 800
MIC= minimum inhibitory concentration; Zone of inhibition = mm; ND= not detected
510

511

512

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