advance phytochemical analysis

1. Submited by:
Mr.Pratik sunil Kapase
(M.Pharm Pharmaceutical Chemistry)
Guided By:
Dr. Smita Pawar
H.O.D. Pharmaceutical Chemistry
PUNE DISTRICT EDUCATION ASSOCIATION’S
Seth Govind Raghunath Sable Pharmacy College, Saswad, Pune

2. Contents:
 Introduction
Classification
Advance process in phytochemicals
Separation and Isolation of Constituent
Tracer Techniques
Various advance process use for phytochemical anlysis

3. INTRODUCTION
Importance of phytochemical analysis
Phytochemical screening searching for bioactive agents
 It refers to extraction ,screening and identification of medicinally active substance s found in
plant
Some of the Bioactive substance that can be derived from plants are flavonoids, alkaloids,
carotenoids, tannins, anti-oxidants and phenolic compounds

4. INTRODUCTION
Analysis of phytochemical properties of medicinal.
The unique biological activity of the plants .
Qualitative and Quantitative analysis of phytochemical can be done using Gas chromatography-
Mass Spectroscopy (GCMS).
GCMS can be applied to solid, liquid and gaseous sample.

5. classification
examples of
phytochemicals
with
supercritical
carbon dioxide
1. Acyl-
phloroglucinols.
2. Alkaloids.
3. Diterpene
4. Fixed oils
5. Pigments
6.Sapogenins

6. Advance process in phytochemicals
Future technologies and development of new techniques and instruments required for modern agro-,
bio-, food and pharmaceutical technologies.
These are closely related with advancements of techniques for desired molecular production and
analytical evaluation required in the industrial process monitoring and quality assessment of raw
materials and products.
A vital aspect of such technologies deals with phytochemicals; therefore, this Special Issue is
devoted to the advancement of the techniques and instruments used for phytochemical analysis that
are currently applied in the a forementioned industries and research and quality assessment
laboratories.

7. Separation and Isolation of Constituent
As the instrumentation for the structure elucidation of organic compounds becomes ever more
effective, and allows the use of increasingly small amounts of material, the most difficult
operation in phytochemical research becomes that of the isolation and purification of plant
constituents.
Although the chemical properties of functional groups and moieties contained in compounds
such as acids, aldehydes, phenols and alkaloids can be exploited for their separation from other
materials, such methods might not fractionate components of the same class; it is in this latter
area that new techniques are constantly being developed.

8. Thin-layer chromatography
Gas–liquid chromatography
Adsorption chromatography
Partition chromatography
Partition chromatography on paper
High-performance liquid chromatography (HPLC)/ high-speed LC
Supercritical fluid chromatography
Electrochromatography
Affinity chromatography
Various methods use for analysis of phytochemical

9. Analysis of the Lithuanian Hops (Hamulus lupulus L.) Varieties by Chromatographic and
Spectrophotometric Methods
Determined chemometric parameters are the following:
(a) the total content of phenolic compounds
(b) the flavonoid compounds
(c) the radical scavenging activity
(d) the qualitative composition of volatile compounds
(e) the total content of xanthohumol in the leaves and cones of Lithuanian hop varieties.

10. Spectrophotometric Analysis of Liquid Extracts
The samples’ total amount of phenolic compounds, flavonoids, and radical scavenging activity
were analyzed using the Folin–Ciocalteu method, colorimetric aluminum chloride, and 2,2-
diphenyl-1-picrylhydrazyl (DPPH) radical methods, respectively, as described in our earlier work
[29]. UV/VIS absorbance was measured using Spectronic 1201 (Milton Roy, Ivyland, PA, USA)
spectrophotometer. Quantitative calibration was performed using rutin in the range of 0.01–
1.00 mg/mL for flavonoids and phenolic compounds, whereas the range of 0.05–0.25 mg/mL
was used for radical scavenging activity. Results were expressed as milligrams of rutin
equivalents (RE) per one gram of dry sample. All measurement results were expressed as
average from three subsequent repetitions.

11. Gas Chromatography-Mass Spectrometry
The analysis of volatile compounds of hop leaves and cones samples after supercritical fluid
extraction was performed using GC-2010 (Shimadzu, Kyoto, Japan) gas chromatography system
with AOC-5000 (Shimadzu, Kyoto, Japan) autoinjector and GCMS-QP2010 mass-spectrometry
detector (Shimadzu, Kyoto, Japan). Separation was carried out using Rtx-5MS (Restek,
Bellefonte, PA, USA) column (30 m × 0.25 mm × 0.25 µm) and helium as the carrier gas. The
analysis was performed under the following conditions: injector temperature 240 ◦C; split ratio
1:10; injection volume 1 µL; and flow rate 1.2 mL/min. The column oven temperature was
programmed from 60 ◦C (held for 3 min), heated up to 150 ◦C at the speed of 2 ◦C/min, held at
150 ◦C for 5 min and then heated to 285 ◦C at the speed of 10 ◦C/min (held for 8 min). Mass
spectrometry detection was performed using electron impact (EI) ionization using 200 ◦C ion
source temperature, 70 eV ionization energy, and 30–400 m/z scan range. Volatile compounds
were identified using NIST05 (NIST, Gaithersburg, MD, USA) mass spectra library and by
calculating RI (retention indices), which were calculated using n-alkanes (C8–C20) retention
times for the same analysis conditions as for the samples

12. High-Performance Liquid Chromatography
HPLC analysis was carried out using a modular system that consisted of solvent reservoirs,
mobile phase pump 9012 (Varian, Palo Alto, CA, USA), mobile phase mixer SP8500 (Spectra-
Physics, Milpitas, CA, USA), Cheminert C1 injector (Valco Instruments, Houston, TX, USA), UV
detector Linear 206 PHD (Linear Instruments, Shefford, UK), Lichrospher 100 RP-18e 5 × 4.0 mm,
5 µm reversed-phase pre-column and column 125 × 4.0 mm, 5µm (Merck, Darmstadt, Germany).
Data was acquired using Clarity Lite Data Acquisition software (Data Apex, Prague, Czech
Republic). Xanthohumol concentration in the methanolic extracts was analyzed according to Kac
and Vovk [15] method with slight modification. The analysis was carried out under isocratic
conditions using solvents A (0.05% trifluoroacetic acid solution in H2O) and B (0.05%
trifluoroacetic acid solution in HPLC grade methanol) at the ratio of 22:78 (A:B). An injection
volume of 20 µL, mobile phase flow rate of 0.7 mL/min, and detection wavelength of 348 nm
was used. The peak of xanthohumol was identified using the retention time of xanthohumol
analytical standard.

13. Hemp Essential Oil Profile and Its
Antimicrobial Activity
GC Analyses of Hemp Essential Oils (EOs) The composition of hemp EOs was characterized by 7890A
gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) coupled to 5975C mass selective
detector (MSD). HP-5ms silica fused capillary column (30 m length × 0.32 mm i.d. × 0.25 µm film
thickness) was used for this purpose. The oven temperature program used was: 40 ◦C for 0min,
increased to 300 ◦C with 5 ◦C/min, held for 10 min. The flow rate of the carrier gas (He) was 1.0
mL/min. The injection volume was 1.0 µL at split ratio 20:1. The temperatures of the ionization source,
the quadrupole and the injector were 230 ◦C, 150 ◦C, and 250 ◦C respectively. The MSD was operated
in full scan mode and all mass spectra were obtained at 70 eV in EI mode. The constituents present in
the EO samples were identified by comparing their linear retention indices (LRI) and MS fragmentation
patterns with those from the NIST008 and Adams mass spectra libraries. The estimated LRI were
determined using a mixture of aliphatic hydrocarbons (C8 to C40) under the same conditions
described above. The GC-FID analysis of the EOs was conducted with a 7890A gas chromatograph
(Agilent Technologies Inc., Santa Clara, CA, USA) coupled to a FID and HP-5 silica fused capillary column
(30 m length × 0.32 mm i.d. 0.25 µm film thickness). The oven temperature was programmed as
mentioned above, whereas the detector and injector temperatures were 280 ◦C and 220 ◦C,
respectively. The carrier gas was helium at a flow rate of 1.0 mL/min. EOs (1.0 µL) were injected using
the split mode. The percentage composition of EO samples was calculated using the peak
normalization method

15. Carotenoid Contents of Lycium barbarum:
A Novel QAMS Analyses, Geographical Origins Discriminant Evaluation, and Storage Stability
Assessment
Instruments and Chromatographic Conditions HPLC-DAD analyses were performed on an
Agilent 1260 Infinity HPLC (Agilent Technologies, Stockport, UK) system (equipped with 1260
Quat Pump VL, 1260 Vialsampler and 1260 DAD WR) by using a column of C30 (YMC, 4.6 × 250
mm, 5 µm).
A gradient of mobile phase was used for efficient separation, mobile phases A
(dichloromethane) and B (methane: acetonitrile: water, 81:14:5, v/v/v), with the elution
program was as follows: 0–20 min, 30% A; 20–48 min, 50% A; 48–50 min, 70% A; 50–55 min,
70%
A. The flow rate was 1 mL/min with the column temperature maintained at 22 ◦C. Detection
wavelength was 450 nm, and the sample injection volume was 20 µL.

16. GC-MS analysis of Nicotiana Flower Concretes
Concretes (50 µL) were diluted in 100 µL of pyridine (Sigma-Aldrich) and 100 µL of N,Obis
(trimethylsilyl)trifluoroacetamide (BSTFA; Supelco, Bellefonte, PA, USA), and incubated at 70 °C for 45 min.
After derivatization, the samples were diluted with 150 µL of chloroform and injected (1 µL) in a system comprised of a
7890A gas chromatograph (Agilent Technologies Inc, Santa Clara, CA, USA) and a 5975C mass selective detector
(Agilent Technologies Inc, Santa Clara, CA, USA).
The column was HP-5 Ms. (30 m × 0.32 mm (i.e.); film thickness 0.25 μm), operated under the following conditions:
Temperature increase from 40 (0 min) to 230 °С at 5 °С/min, held at 230 °С for 10 min; injector and detector
temperatures of 250 °С; helium as a carrier gas at a 1 mL/min constant flow rate; mass detector scan range m/z = 50–550;
split mode (5:1).

17. Identification of the detected compounds was carried out using mass spectra library data ([53], NIST 08 database; own
libraries). Calculation of the retention (Kovat′s) indices was done using a calibration mixture of n-alkanes (C8–C40) in n-
hexane.
The content of the identified compounds was presented as a percentage of the total ion current (TIC), following the
normalization method of the recorded peak areas.

18. Statistics
 All data were presented as mean value ± standard deviation, resulting from a threefold repetition of experiments. Statistical significance of
differences was assessed by ANOVA and Tukey’s multiple comparison test (p < 0.05).
Conclusions
The GC-MS analysis of the concretes obtained through extraction with n-hexane from the fresh flowers of four Nicotiana species, N.
rustica, N. glutinosa, N. alata, and N. tabacum, identified their major and minor constituents.
Differences were observed between the species, as well as between the genotypes studied. The characteristic olfactory profiles of the
concretes and their sufficient yields are a prerequisite for the possible processing of Nicotiana flowers and the obtaining of new aromatic
products with use in perfumery and cosmetics.
These are the first results, which characterize the flowers of the regarded Nicotiana species as potent sources for obtaining these natural
aromatic products

19. (UMSF) as a Powerful Tool for Bioactive Molecules Discovery.
 Assay guided fractionation, a directed and iterative process of chemical extraction, purification, and bioassay, has
arguably been the paradigm technique for pharmacognosy's across decades of study.
Indeed, the plant-derived chemotherapy agents paclitaxel, vinca alkaloids, and camptothecin , were all discovered using
this approach, ultimately saving thousands of lives through applied clinical practice.
Despite the rise and fall of synthetic combinatorial chemistry in pharmaceutical drug development, assay-guided
fractionation has remained an indisputable tool in bioactive molecules discovery.
Initially, open-column chromatographic separations were predominantly utilized in assay guided fractionation, typically
performed using a silica gel particle stationary phase.
By today’s standards, these separations were largely inefficient, requiring copious volumes of volatile solvents and large

20. Analysis of Lithuanian Hops (Humulus lupulus L.) Varieties by
Chromatographic and Spectrophotometric Methods
Gas Chromatography-Mass Spectrometry
The analysis of volatile compounds of hop leaves and cones samples after supercritical fluid extraction was performed using
GC-2010 (Shimadzu, Kyoto, Japan) gas chromatography system with AOC-5000 (Shimadzu, Kyoto, Japan) autoinjector and
GCMS-QP2010 mass-spectrometry detector (Shimadzu, Kyoto, Japan).
Separation was carried out using Rtx-5MS (Restek, Bellefonte, PA, USA) column (30 m × 0.25 mm × 0.25 µm) and helium
as the carrier gas.
The analysis was performed under the following conditions: injector temperature 240 ◦C; split ratio 1:10; injection volume 1
µL; and flow rate 1.2 mL/min.
The column oven temperature was programmed from 60 ◦C (held for 3 min), heated up to 150 ◦C at the speed of 2 ◦C/min,
held at 150 ◦C for 5 min and then heated to 285 ◦C at the speed of 10 ◦C/min (held for 8 min).

21. Mass spectrometry detection was performed using electron impact (EI) ionization using 200 ◦C
ion source temperature, 70 eV ionization energy, and 30–400 m/z scan range.
Volatile compounds were identified using NIST05 (NIST, Gaithersburg, MD, USA) mass spectra
library and by calculating RI (retention indices), which were calculated using n-alkanes (C8–C20)
retention times for the same analysis conditions as for the samples.

22. High-Performance Liquid Chromatography
 HPLC analysis was carried out using a modular system that consisted of solvent reservoirs, mobile phase pump 9012 (Varian, Palo Alto,
CA, USA), mobile phase mixer SP8500 (Spectra-Physics, Milpitas, CA, USA), Cheminert C1 injector (Valco Instruments, Houston, TX,
USA), UV detector Linear 206 PHD (Linear Instruments, Shefford, UK), Lichrospher 100 RP-18e 5 × 4.0 mm, 5 µm reversed-phase pre-
column and column 125 × 4.0 mm, 5µm (Merck, Darmstadt, Germany).
Data was acquired using Clarity Lite Data Acquisition software (Data Apex, Prague, Czech Republic). Xanthohumol concentration in the
methanolic extracts was analyzed according to Kac and Vovk [15] method with slight modification.
The analysis was carried out under isocratic conditions using solvents A (0.05% trifluoroacetic acid solution in H2O) and B (0.05%
trifluoroacetic acid solution in HPLC grade methanol) at the ratio of 22:78 (A:B).
An injection volume of 20 µL, mobile phase flow rate of 0.7 mL/min, and detection wavelength of 348 nm was used. The peak of
xanthohumol was identified using the retention time of xanthohumol analytical standard.

23. Conclusions
Adetailed phytochemical composition analysis of the leaves and cones of five Lithuanian hop
varieties was carried out. The findings are the following: (a) The QUENCHER procedure shows
1.2–3.5 times more total content of phenolic compounds than the classical method with
conventional solvent extraction. (b) The QUENCHER procedure reveals matrix
encapsulated/adsorbed/insoluble substances. (c) The leaves of the hops contain phenolic
compounds that are insoluble in aqueous 75% methanol. (d) The content of phenolic
compounds and antioxidant activity correlates. (e) The highest value of phenolic compounds and
radical scavenging activity was in the suspensions of leaves and cones of Fredos taurieji’ and
‘Fredos derlingieji’. (f) The highest concentration of xanthohumol in the leaves was 0.0080%, and
in the cones was 0.2136% of dry mass. (g) The xanthohumol content in hop cones strongly
correlates with flavonoid, bitter α-acids, and β-acids contents. (h) The highest amounts of hop’s
bitter acids were determined in ‘Kauno gražieji’ and ‘Raudoniai’. ‘Raudoniai’ leaves were
exceptional in their aroma. The study shows that further investigation and valorization of
different hop biomass components is of importance, especially the potential of leaves and
wines.

24. Promising Endophytic Alternaria alternata from Leaves
of Ziziphus spina-Christi: Phytochemical Analyses,
Antimicrobial and Antioxidant Activities
Gas Chromatography-Mass Spectrometry (GC–MS)
Analysis EA crude extract of A. Alternaria was injected to GC–MS to identify the metabolic
compounds. GC–MS analysis was achieved using Agilent Technologies GC–MS 5977A operating at
70 eV and computer mass spectral library (NIST, 2011 version). The spectrum of the unknown
constituents was matching with the available data stored in GC–MS libraries.
Transmission Electron Microscopy (TEM) In order to study the efect of EA crude extract of A. alternata
on ultrastructure of the most sensitive bacteria, bacterial cells were collected by centrifugation at
4000 rpm for 10 min from 24-h old cultures grown on nutrient broth media and washed with distilled
water; the samples were fxed in 3% glutaraldehyde, rinsed in phosphate bufer, and post-fxed in
potassium permanganate solution for 5 min at room temperature. The samples were dehydrated in
an ethanol series ranging from 10 to 90% for 15 min in each alcohol dilution and fnally with absolute
ethanol for 30 min. Samples were infltrated with epoxy resin and acetone through a graded series
until fnally in pure resin. Ultrathin sections were collected on copper grids. Sections were then double
stained in uranyl acetate followed by lead citrate. Stained sections were observed with a JEOL—JEM
1010 TEM at 80 kV at RCMB, Al-Azhar University

26. Conclusions
In the present study, a new QAMS method was established by using trans-β-apo8 0 -carotenal, a
substance that did not exist in the LB sample, as a standard reference to determine the four
carotenoids in LB simultaneously. The small SMD between ESM and QAMS methods implied the
feasibility of our method. By establishing the RCF between the standard reference and
carotenoids, the quantities of the carotenoids can be directly calculated in practical applications,
thus the QAMS method provided a more convenient, faster, cheaper, and simpler way for
evaluating the quality of LB. Based on the carotenoid content, 34 batches of LB samples could be
clearly divided into two groups by HCA, PCA, and OPLS-DA analysis (Group 1: Qinghai; Group 2:
Ningxia and Gansu), which was directly related to the geographic locations of the different LB
samples. The storage stability test of LB implied that zeaxanthin dipalmitate content decreased
significantly as the color of LB changed from bright red to black-brown under high humidity and
high temperature conditions, even at the ambient temperature.