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\begin{document}

\title{Antihypertensive and Hepatoprotective activity of optimized eupalitin
3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside from Boerhavia diffusa Linn.\selectlanguage{english}}



\author[1]{Vidhu  aeri}%
\author[2]{ILYAS UK}%
\affil[1]{Jamia Hamdard}%
\affil[2]{Moulana College of Pharmacy}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Boerhavia diffusa is an herbaceous member of the
Nyctaginaceae family which has been widely used in folk medicine to
treat several illnesses. Experimental Approach: A validated HPTLC method
has been newly developed for the quantification of eupalitin 3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in hydroalcoholic extracts of B. diffusa. A
three-level factor Box-Behnken statical design was used for
optimization, extraction time (min), temperature (\selectlanguage{ngerman}ºC), and methanol:
water ratio (\% v/v) are independent variables while bioactive compounds
as the dependent variable. This study was aimed to investigate the
pretreatment of HepG2 cells with hepatoprotective agents against the
damage induced by galactosamine (GalN) and evaluate in vivo
antihypertensive activity of eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in
rats by prednisolone inducing hypertension. Key Results: The separation
was achieved on silica gel 60F254 HPTLC plate using: toluene: acetone:
water (5: 15:1) as mobile phase. Densitometric analysis of eupalitin
3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside was carried out in the absorbance mode at 366
nm. A study has shown that optimized eupalitin 3-O-\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside
can be used as an antihypertensive drug in rats by prednisolone inducing
hypertension and In-vitro Hepatoprotective Activity of optimized
eupalitin-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside was checked in HPG2 cell line
compared with silymarin by ANOVA technique. Conclusion and Implications:
It is concluded that A new method for a method of validation and
quantification of eupalitin-O-\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in Boerhavia diffusa
has been developed, In vivo antihypertensive and in-vitro
hepatoprotective activity of optimized eupalitin O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside was done in intoxicated rats and in HepG2 cells
induced by galactosamine (GalN).%
\end{abstract}%



\sloppy


\textbf{Introduction}

The inflammation of hepatitis is swelling that occurs when tissues of
the body are infected or injured. Hepatitis is characterised as acute or
chronic, depending on how long the inflammation lasts and how much the
hepatic parenchyma is damaged {[}1{]}. Viruses are classified into five
types: A, B, C, D, and E. Because of the burden of illness and
mortality, these five types are the most concerning. The disorder might
be self-limiting (i.e., it will go away on its own) or it can develop to
fibrosis (scarring) and cirrhosis {[}2{]}. Parasites and viral
infections, autoimmune illnesses, and intoxication with xenobiotics such
as alcohol, herbal medicine, pharmaceuticals, chlorinated solvents,
peroxidized fatty acids, fungal toxins, industrial pollutants, and
radioactive isotopes are all examples of hepatic problems.Types A and C,
in particular, cause chronic disease in hundreds of millions of people
and are the leading causes of liver cirrhosis and cancer {[}3{]}. In
India, medicinal herbs are a key source of hepatoprotective drugs. More
than 700 mono and polyherbal preparations in the form of decoctions,
tinctures, and pills have been used for various liver ailments {[}4{]}.
Ayurveda is the earliest Indian system of medicine, defining many plants
for the management of hepatotoxicity. Since times plants have been used
therapeutically in variety of conditions and by the arrival of current
synthetic medicines and their accessibility of consistent dosage forms,
usage ease and theatrical efficiency in acute circumstances, but there
was decrease in the usage of medicinal plant till herbal rebellion
{[}5{]}.~ As compared to the narrow spectrum effect of synthetic drugs
with the associated danger of side effects, traditional drugs show minor
action and require long term administration to be effective, mainly in
chronic conditions {[}6{]}. Many plant-based medications, such as
Andrographis paniculata, Boerrhavia diffusa, Calotropis
procera,\emph{Fumaria indica, Garcinia cambogia, Luffa acutangula,
Mamordic subangulata, Naragamia alata, Nigella sativa,} and
\emph{Trigonella foenum graecum} have been proven to be hepatoprotective
in nature. {[}7{]}\emph{.} Hepatitis and jaundice are major liver
ailments, with an elevated toll of mortality. At present, only rare
hepatoprotective allopathic medicines are accessible for the management
of liver ailments. As a result, people are adapting various plant
extracts for the treatment of liver syndromes. From both a morphological
and functional standpoint, the galactosamine-induced experimental model
system in rats is acknowledged to be similar to viral hepatitis in
humans. Galactosamine has a high liver specificity compared to other
dangerous groups such as acetaminophen, carbon tetrachloride,
paracetamol and so on, because hepatocytes have high levels of
galactose-1-uridyltransferase, galactokinase and galactosamine does not
harm other organs. Hepatotocity is caused by galactosamine, which causes
patchy hepatocyte necrosis and significant portal and parenchymal
infiltration (4). By boosting the formation of UDP-sugar derivates,
galactosamine causes uridine diphosphate (UDP) depletion, which causes
inhibition of RNA and protein synthesis, leading to cell membrane
deterioration {[}8{]}. This study was aimed at investigating the
pretreatment of HepG2 cells with hepatoprotective agents against the
damage induced by galactosamine (GalN). Hypertension is a serious
medical chronic illness that gradually increases the risk of kidney,
heart, brain, and other illnesses. Hypertension is a major cause of
death that takes place before the average age of death in a certain
population worldwide. Hypertension affects an estimated 1.28 billion
people globally, the majority of whom live in middle and low-income
nations. It increases the risk of cardiovascular diseases, including
diffuse atherosclerosis, heart failure, dementia, and cognitive
impairment. Synthetically, antihypertensive drugs have been suggested to
treat hypertension. However, the therapeutic activity of these drugs is
low and also generates various side effects, which involve visual
disorders, dry mouth, dizziness, emotional distress, gastrointestinal
disturbances, etc. Therefore, it requires searching for cheaper and non-
toxic natural products. Plants are generally used in the traditional
system of medicine for the treatment of several diseases. In the past
few decades, a lot of investigation has been done on local medicinal
plants for hypotensive and antihypertensive potential. Plants are a rich
source of flavonoid glycosides, which may have antihypertensive
potential. The present investigation is focused on the effect of the
anti-hypertensive activity of flavonoids and glycosides on rat induced
methylprednisolne hypertension. \emph{Boerhavia diffusa is a herbaceous
Nyctaginaceae family member with a long history of use by Brazil's
indigenous and tribal peoples.} In particular, the roots and leaves of
this plant have been widely used in folk medicine to treat several
illnesses, including those affecting the gastrointestinal tract. This
plant has been reported for behavioural and neuroendocrine effects
{[}1{]}, antidepressant activity {[}2{]}, anti-angiogenic activity
{[}3{]}, anticonvulsant and antiepileptic {[}4{]}, anti-inflammatory
activity {[}5{]}, prostatic hyperplasia {[}6{]}, genotoxic and
antigenotoxic {[}7{]}, thrombolytic, cytotoxic and antimicrobial
activities {[}8{]}. arsenic trioxide induced cardiotoxicity {[}9{]},
antiurolithic activity {[}10{]}, anti-hyperglycaemic and reno-protective
effect {[}11{]}, acetaminophen induced liver toxicity studies {[}12{]} ,
antiproliferative and antiestrogenic effects {[}13{]},~
immunomodulatory, anti-metastatic activity {[}15{]} , radio-protective
activity {[}16{]}, potent anti-breast cancer activity {[}17{]},
intestinal activity {[}18{]}, immunosuppressive activity {[}19{]},
anti-diabetic activity {[}20{]}, ~ CA2+ channel antagonistic {[}21{]},
~chemo-preventive action {[}22{]}, glycol-induced urolithiasis {[}23{]},
and inhibition of~ human cervical cancer cells {[}24{]}.~ Several
flavonoid glycosides are isolated from the leaves of this plant that
show potent anticancer, powerful antioxidant and geno-protective
properties, intestinal motility, and spasmolytic activity {[}25{]}. The
objective of the study was to optimize, validate and quantify eupalitin
3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in hydroalcoholic extracts of B. diffusa~and
also investigate hepatoprotective and antihypertensive activity of
isolated eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside.

\textbf{Materials and Methods}

\textbf{Chemicals and reagents}

Isolated and purified of eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside from
Hamdard laboratory, New Delhi, India. Methanol HPLC grade (SD Fine
Chemicals, Mumbai, India) was used as a solvent for the preparation of
standards and samples. Toluene, ethyl acetate, formic acid, and methanol
(CDH Labs, Mumbai, India) were used as mobile phases for HPTLC analysis.
2- Amino ethyl diphenylborinate (Sigma-Aldrich Co. LLC, St. Louis, MO,
USA). All solutions used for the analysis were filtered through a 0.22
\selectlanguage{greek}μ\selectlanguage{english}m Syringe- driven Filter (HIMEDIA, Mumbai, India). Dulbecco's modified
eagle's medium (DMEM), foetal bovine serum (FBS), HepG2 cell line
(National Centre for Cell Science (NCCS), Pune, India), MTT test kit,
trypsin EDTA, Trypan blue solution, and 100\% ethanol (Himedia Lab Pvt.
Ltd., Mumbai, India), Penicillin and Streptomycin, and DMSO
(Sigma-Aldrich Co. LLC, St. Louis, MO, USA).

\textbf{Instruments}

Plant material extracts were created by hot percolation using Soxhlet
extractor (Omega, Mumbai, India). Extracts were dried underneath vacuum
on Rotary evaporator (Buchi R-114, Switzerland). For HPTLC analysis of
bioactive compounds, a cAMAG HPTLC system (Muttenz, Switzerland)
equipped with a Linomat IV sample applicator was used. Extracts were
applied on aluminum- backed TLC plates (10 \selectlanguage{ngerman}× 20 cm) pre-coated with
silica gel 60F\textsubscript{254} (Merck, Darmstadt, Germany). For
developing the TLC plates, a CAMAG twin trough chamber (vertical
development) was used. Tissue culture flasks, 96 and 24 well micro
culture plates, eppendorf tube, inverted microscope, serological
pipette, heamocytometer (Himedia Lab Pvt. Ltd., Mumbai,
India)\textbf{,}Laminar flow hoods (Khera instrument, New Delhi, India),
CO\textsubscript{2} incubator (NuAire, USA), Water bath, Deep freezer
(-20°C).

\selectlanguage{greek}\textbf{Isolation of eupalitin 3-O- β-D-galactopyranoside}\selectlanguage{english}

Dried powdered plant material was subjected to extraction by using
methanol for 6 h at 37\selectlanguage{ngerman}°C respectively. The combined hydro alcoholic
extracts were pooled and evaporated to dryness under reduced pressure at
40 ºC with rotator evaporator. Extracts was filtered and concentrated in
rotary evaporator under reduced pressure to get residues (28\%). The
crude extracts obtained were portioned-fractionated with hexane (3×250
ml), chloroform (3×250 ml) and ethyl acetate (3×250 ml). The dried ethyl
acetate recidue (15 g) was thoroughly mixed in hot methanol and stored
overnight in the refrigerator, filtered, the solid seperated,
cristallized with hot methanol, to obtain eupalitin 3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside (0.1578\%w/w).

\textbf{Preparation of Sample Solutions}

Accurately weighed 100 mg of crude extracts were dissolved in 10ml of
HPLC grade of methanol and sonicated the solution for 10 min and then
makeup with 10 ml methanol; filter the solution through Before injecting
into the HPTLC system, use a 0.22 syringe filter.

\textbf{Preparation of Standard solutions}

The standard solution of bioactive compounds was prepared by dissolving
accurately weighed 1.0 mg of Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in 1.0
ml of methanol HPLC grade as stock solution and stored at 4@\selectlanguage{english}C. These
standards were further diluted as per the requirement to plot a desired
concentration for quantification.

\textbf{Calibration curves for standard markers}

Standard solution of Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside was applied in
triplicate silica gel 60 F\textsubscript{254} plates, using a CAMMAG
Linomat-5 Automatic Sample Spotter. The plates were developed in solvent
toluene: acetone: water (5: 15:1 v/v) in a CAMAG glass twin trough
chamber (20\selectlanguage{ngerman}×10 cm) up to a space of 8 cm. After development, the plates
were dried in air and scanned at 254 nm using CAMAG TLC scanner 3 and
win CATS 4 software. The peak areas were recorded. Calibration curves of
Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside were obtained by plotting peak
areas versus applied crude extracts concentration of Eupalitin 3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside respectively.

\textbf{Estimation of different markers}

With a CAMAG Linomat-5 Automatic Sample Spotter, ten microliters of each
of the sample solutions were applied in triplicate on silica gel 60 F254
plates. The peak areas and absorption spectra were recorded. The number
of bioactive compounds in \emph{B. diffusa} was calculated using the
respective standard calibration curves of Eupalitin 3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside

\textbf{Limits of detection and quantification}

The lowest amount of analyte in a sample that can be detected but not
necessarily quantified is known as the limit of detection. The lowest
amount of analyte in a sample that can be quantified with sufficient
precision and accuracy is the limit of quantitation. The quantification
and detection limits were determined using the signal-to-noise ratio
approach using Eqns. (1) and (2).

LOQ =10 \selectlanguage{greek}σ/ \selectlanguage{english}S\ldots{}\ldots{}\ldots{}\ldots{}\ldots{} Eq. (A.1)

LOD = 3.3 \selectlanguage{greek}σ/ \selectlanguage{english}S\ldots{}\ldots{}\ldots{}\ldots{}\ldots{} Eq. (A. 2)

Where S is the slope of the calibration curve and is the standard
deviation of the intercept of the calibration plot.

\textbf{Precision}

The determination of was done using intra-day (repeatability) and
inter-day (intermediate precision) fluctuations at three different
concentration levels of 100, 200, and 400ng/band. The tests were carried
out in three different ways.

\textbf{Specificity}

The method's specificity was determined by examining standard
medications and samples. By comparing the Rf values and spectra of the
band with Eupalitin 3-O---D-galactopyranoside, the band for alco-holic,
hydroalcoholic, aqueous extracts was confirmed. The peak purity of
standards was determined by comparing the spectra at three different
levels: peak start, peak apex, and peak-end position.

\textbf{Accuracy in the context of recovery}

Recovery trials utilising the usual addition method were used to
determine accuracy. The combination was spiked with 50, 100, and 150
percent of Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside before being tested.

\textbf{Optimization of bioactive compounds in hydroalcoholic extracts}

A box-Behnken applied math style with three factors, 3 levels, and
seventeen runs was chosen for the optimisation study using the
Design-Expert V6 Software package (Stat-Ease Inc., Minneapolis, MN,
USA). This style is appropriate for exploring quadratic response
surfaces and constructing second-order polynomial models. The
experimental style consists of a group of purposes lying at the center
of every edge and also the replicated center point of the
four-dimensional cube that defines the region of interest. The dependent
variables and independent are listed in Table 1.

The polynomial equation generated by this experimental design equation

R = C\textsubscript{0} + C\textsubscript{1} X\textsubscript{1} +
C\textsubscript{2} X\textsubscript{2} +
C\textsubscript{3}X\textsubscript{3} + C\textsubscript{4}
X\textsubscript{1}X\textsubscript{2} + C\textsubscript{5}
X\textsubscript{1}X\textsubscript{3} + C\textsubscript{6}
X\textsubscript{2}X\textsubscript{3} +
C\textsubscript{7}X\textsubscript{1}\textsuperscript{2} +
C\textsubscript{8}X\textsubscript{2}\textsuperscript{2} +
C\textsubscript{9}X\textsubscript{3}\textsuperscript{2} \ldots{}. Eq.
(A. 3)

Where R is the dependent variable, C\textsubscript{0} is the intercept,
C\textsubscript{1} to C\textsubscript{9} are the regression
coefficients, and X\textsubscript{1,} X\textsubscript{2} and
X\textsubscript{3} are the independent variables.

Table 2 shows the experimental style matrix. The data obtained from the
planning was evaluated by using the Design-Expert software system.
Numerical improvement was meted out for unveiling the optimized
bioactive compounds in aqueous alcohol. The optimized eupalitin 3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in aqueous alcohol was ready and evaluated.

\selectlanguage{greek}\textbf{Optimized Eupalitin 3-O- β-D-galactopyranoside's
hepatoprotective efficacy against D-galactosamine-induced toxicity}\selectlanguage{english}

\textbf{Maintenance of cell lines:}

HepG2 cell line were grown in 25 cm\textsuperscript{2} tissue culture
flasks containing minimum essential medium (MEM) supplemented with 10\%
FBS, 1\% penicillin /streptomycin at 37\selectlanguage{ngerman}°C in a
CO\textsubscript{2}incubator in an atmosphere of humidified 5\%
CO\textsubscript{2} and 95\% air. The cells were kept alive by
sub-culturing them in 25 cm\textsuperscript{2} tissue culture flasks on
a regular basis.

\textbf{Method for passaging the cells}

Before using, all the reagents were brought to room temperature. A 10 ml
serological pipette was used to remove media from the 80-90 percent
confluent flasks. 10 mL PBS was used to wash the cells in the T-75
flask. The flask was filled with two millilitres of 0.1 percent trypsin
EDTA. The flask was kept at 37°C in the CO\textsubscript{2} incubator
for 2-3 min and was observed under microscope for detachment. For
trypsin inhibition, six millilitres of growth media were added to the
flask and pipetted back into place. The cell suspension was collected in
15 ml falcon tube, and then centrifuged at 1200 rpm for 3 min.
The~pellet~was~resuspended~in~3~ml~of~complete~media~after~the~supernatant~was~discarded.
Cells were counted, and then 0.2-0.4 million cells were kept in T-25
flask for growing. The flasks were incubated in
CO\textsubscript{2}incubator at 37 °C and the cells were periodically
monitored for any morphological changes and contamination. After the
formation of 80- 90\% confluent monolayer, the cells were further
utilized.

\textbf{Procedure:}

Cells were seeded at a density of 1×10\textsuperscript{5}cells/ in each
well of the 24 well plates and incubated overnight. After 24h, the media
was flicked off, the cells were treated with eupalitin 3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside (100 and 500 \selectlanguage{greek}µ\selectlanguage{english}g/ml) in separate wells of a 24-well
plate and incubated for 2 h. Silymarin (250, 500\selectlanguage{greek}µ\selectlanguage{english}g/ml) was used as a
reference standard After incubation, the cells were treated with
D-galactosamine at a concentration of 40mM (Quintero et al 2002) and was
allowed to incubate for 2h. After incubation , the cells were washed and
incubated for 1 h with MTT (20 \selectlanguage{greek}μ\selectlanguage{english}l of 5mg/ml of MTT in PBS) was added to
each well. Formation of formazan crystal was observed under a
microscope. In case crystal, formation was not proper, incubation was
continued for an hour more. Media was removed and the remaining formazan
crystals were dissolved in 200 \selectlanguage{greek}μ\selectlanguage{english}l of DMSO in each well. The cell culture
plate was kept on a shaker for 15 min; The absorbance was recorded by
ELISA reader at 540 nm.

The \% hepatoprotection was obtained by the following formula:

\% hepatoprotection =100 - {[}((absorbance of control - absorbance of
test)/absorbance of

control)\selectlanguage{ngerman}×100

\selectlanguage{greek}\textbf{In-Vivo Antihypertensive Activity of isolated eupalitin 3-O-
β-D-galactopyranoside}\selectlanguage{english}

\textbf{Identification, Procurement and housing of animals}

Albino rats (body weight 100-200 g) were supplied by Central Animal
House facility (Registration Number: 173/CPCSEA and Date of
Registration: 28, JAN-2011), Jamia Hamdard and kept under standard
laboratory conditions in 12 h light/dark cycle at 25 0C \selectlanguage{ngerman}± 2 0C. Pellet
feed (Lipton, Calcutta, India) and unlimited water ad libitum~was given
to the animals. They were labelled to make it easier to identify them.

\textbf{Animals} \textbf{training and conditioning}

The animals were restrained in a restrainer for 10 min every day for one
week in order to conduct the BP measurement tests. This exercise was
performed to reduce blood pressure fluctuation caused by the animal's
behaviour while in the restrainer for activity measurement.

\textbf{Hypertension induction in normotensive rats}

After recording the initial BP of rats, the animals were divided into
groups of 4 animals each. One group was chosen as the control group. The
remaining groups were given a subcutaneous injection of methyl
prednisolone acetate (20 mg/kg/wk) for 2 weeks, as described by Krakoff
et al.\textsuperscript{24}

\textbf{Rats' mean blood pressure was measured.}

The CODA Non Invasive Blood Pressure Recorder was used to measure mean
arterial blood pressure in conscious rats using the Tail-Cuff method
(Kent Scientific Corporation, USA). With the rat's tail protruding, the
restrainer was inserted in the BP instrument. The rat's restrainer was
placed in the BP instrument with its tail protruding. The tail was
gently pressed against a transducer mem-brane that was linked to the
digital BP display panel. After that, the device was turned on and
allowed to stabilise until a consistent pulse rate was seen. The BP
recording button was pressed and the mean arterial BP was recorded after
the ''pulse level ready'' indicator showed. In this investigation,
albino rats (body weight 150-200 g) were employed. Rats were divided
into four groups, each with four individuals. After suspension in 0.1
carboxy methyl cellulose (CMC) solution, each drug (10, 20, 40, and 60
mg/kg body weight) was injected intraperitoneally. After 1 hour, the
mean arterial blood pressure was measured.

\textbf{Statistical analysis of data:}

GRAPHAD INSTAT 3 was used to do the statistical analysis (Graph Pad
Software Inc, San Diego, CA). The results of animal trials were
expressed as the arithmetic mean minus the standard error of the mean
(SEM). One-way analysis of variance (ANOVA) was used to assess the
effects of the various groups, and Dunnet's multiple comparison tests
were used to compare the effects of the treatment groups to the toxic
control group. The significance level was set at p0.01. The MABP
reduction \% for each therapy group was also calculated and compared.

\textbf{Results and Discussion}

\textbf{The HPTLC approach was used to develop and validate bioactive
marker.}

\textbf{Optimal mobile phase development}

The working standard solution of Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside
(1m/ml) in methanol was used for chromatographic separation
investigations. Initially, multiple solvent solutions were used in
various trials. Finally: toluene: acetone: water (5: 15:1) for
determination of Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside gave a sharp and
well-defined peak. We obtained well-defined bands upon saturating the
chamber with the mobile fraction at room temperature for 30 min. The
standard band of Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside along with
hydroalcoholic extracts of \emph{B. diffusa} presented on HPTLC plate
scanned at 366 nm (Fig. 1).

\textbf{LOD and LOQ}

The LOD and LOQ were determined on the basis of three and seven times
the noise levels, respectively. The results listed in Table 3

\textbf{Precision}

Subheadings may be used to split this section. It should give a clear
and succinct summary of the experimental findings, their interpretation,
and the possible experimental inferences.The repeatability of
measurement (n = 6) of peak area for Eupalitin 3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside were expressed in terms of percent CV (\% RSD).
The intra-day and inter-day variation for the determination of was
carried out at three concentration levels; 100, 200, and 400ng/band. The
results depicted in Table 3 showed that no significant intra and
inter-day variation was obtained in the analysis of bioactive compounds

\textbf{Specificity}

The specificity of the developed method for the analysis of Eupalitin
3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in the extracts was ascertained by analysis
of standard and sample. The six tested polyphenols in samples were
confirmed by comparing the R\textsubscript{f} value and spectra of the
spot with that of the standard. The peak purity of individual compounds
was assessed by analyzing the spectra at peak start, peak apex, and peak
end positions of the band.

\textbf{Accuracy as recovery}

Accuracy was investigated by analyzing three concentrations of standard
drug solution previously analyzed using the standard addition technique.
The recovery studies were carried out to check the sensitivity of the
method to estimate eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside. The standard
addition technique was carried out by adding 50, 100 and 150\% of the
bioactive compound's concentration in the sample extracts. The \%
average recoveries of the concentrations were found to be 99.8\% for
eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside indicative of high accuracy. The
value of \% average recovery is displayed in Table 3. The mean \%
recovery values, close to 100\%, and their low \% RSD values indicated a
high accuracy of the analytical method.

\textbf{Box Behnken experimental design}

Using seventeen experimental trial runs, a three-level factorial, Box
Behnken applied statistical experimental approach was performed. Table 2
lists the independent variables as well as the answers for all optimum
trial experimental runs. The quadratic model was determined to be the
best-fitting model for standard Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside,
and the comparative values of R, SD, and percent CV for the various
planned models are presented in Table 4 along with the regression
equations normally for eventually elite replies. Only statically
significant (P 0.0001) coefficients for Eupalitin 3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside are included in the equations. A positive number
implies that the effect favors the optimization, whereas a negative
value shows that the factor and the response have an inverse connection.
The equations show that time (X1), temperature (X2), and solvent ratio
(X3) have a negative effect on the reaction, while time (X1),
temperature (X2), and solvent ratio (X3) have a positive effect (Y1). It
also demonstrates that the relationship between reaction and variables
isn't always straightforward. A factor can create a variable degree of
response when more than one factor is modified at the same time. When X1
and X2, as well as X1 and X3, interact, the reaction is negatively
impacted. However, in the case of the square root of many components,
the outcome does not repeat history, as demonstrated by its performance.
In the instance of the square root of variables,
X\textsuperscript{2}\textsubscript{2} and
X\textsuperscript{2}\textsubscript{3} showing positive and
X\textsuperscript{2}\textsubscript{1} X22 and X23 had a positive impact
on the replies in Table 4, but X21 had a negative impact. The final
compositional ratios of the extractions were selected on the basis of
percentage yield of Eupalitin 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside using 3
D-response surface plots (Fig. 2).

\selectlanguage{greek}\textbf{Effect of independent factors on Eupalitin 3-O-
β-D-galactopyranoside (Y\textsubscript{1})}\selectlanguage{english}

Regression equation of the fitted models:Y\textsubscript{1}=
0.035-0.00097 X\textsubscript{1} + 0.006 X\textsubscript{2} + 0.0031
X\textsubscript{3} + 0.0038 X\textsubscript{1} X\textsubscript{2} +
0.0043 X\textsubscript{1} X\textsubscript{3} - 0.0051
X\textsubscript{2}X\textsubscript{3} The extraction time
(X\textsubscript{1}) affected on the \% yield of eupalitin. It has
observed from the equation X1 have negative effect on eupalitin as
compared to X\textsubscript{2} and X\textsubscript{3}. When increase
time of extraction increases, the \% yield of eupalitin decreases due to
degradation of compound as shown figure in (Fig. 2A). Factor
X\textsubscript{2} (temperature) have prominent positive effect on \%
yield of eupalitin than factor X\textsubscript{1} and
X\textsubscript{3}. As the temperature increased, the \% yield of
eupalitin also increased respectively due to thermo stable compound as
shown in (Fig. 2B). Factor X\textsubscript{3}(solvent ratio) showed less
positive effect on eupalitin percentage yield as compared to that
observed on factor X\textsubscript{2}. It was observed that, when higher
level of solvent ratio was increased, \% yield of eupalitin also
increased because medium polar compound as shown figure in (Fig. 2C).

\selectlanguage{greek}\textbf{Identification of eupalitin- 3-O- β-D-galactopyranoside}\selectlanguage{english}

The compound appareled as dull yellow florescent under UV light because
a flavone with free hydroxyl group at 5\textsuperscript{th} position.
The compound's EIMS revealed a molecular ion peak at m/z 184(A+) and
121(B+), respectively, indicating the presence of one hydroxyl in the
b-ring and~two methoxyl groups, one hydroxyl in the a-ring . The
aromatic region of 1H NMR (in DMSO-d6, 400 MHz) revealed three signals
at \selectlanguage{greek}δ\selectlanguage{english}~6.40 (1H,s),~ \selectlanguage{greek}δ\selectlanguage{english}6.94(2H,d, J=8.5 Hz), and~ \selectlanguage{greek}δ\selectlanguage{english}8.06(2H,d, J=8.5 Hz),
which were assigned to H-8,H-31/5', and H-2'/6', respectively. Two
signals in the aliphatic region at \selectlanguage{greek}δ\selectlanguage{english}3.76 (3H,s) and \selectlanguage{greek}δ\selectlanguage{english}3.87(3H,s) were
identified to --OCH3 groups at C-6 and C-7 position present in the
A-ring. Thus compound has been deduced to be 4', 5-dihydroxyl-6,7-
Figure 2: \textsuperscript{1}HNMR spectra of eupalitin-3-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside dimethoxyflavonol or eupalitin.

\selectlanguage{greek}\textbf{Effect of eupalitin- 3-O- β-D-galactopyranoside on galactosamine
induced hepatotoxicity}\selectlanguage{english}

In the study, we examined the possible hepatoprotective activity of
purified eupalitin, remain ethyl acetate fraction and chloroform
fraction against GalN-induced cytotoxicity, by pre-incubating the cells
with or without the extracts or sylimarin. A significant decrease in
cell viability was observed upon treatment of HepG2 cells with GalN
(4Mm).Treatment with purified eupalitin and other fractions demonstrated
a significant dose-depended protection toward cell toxicity resulting
from GalN exposure. Results are expressed as mean \selectlanguage{ngerman}± standard error mean
(S.E.M). From graph plotting, percentage protection against sample
concentration, a sample concentration providing percentage
cytoprotection of standard silymarin (84 percent at 500g/ml), purified
eupalitin (67 percent at 1000g/ml), chloroform fraction (23 percent at
1000g/ml) and remain ethyl acetate fraction (42 percent at 500g/ml) was
calculated. On Fig .3 and Table 5, the \% protection is graphically
represented. The experiment was carried out three times. Graphpad prism
5.0 was used for statistical analysis, which included Dunnett's multiple
comparison tests and one-way analysis of variance (ANOVA) (Graphpad
Software, Inc., San Diego, CA, USA).

\textbf{Optimized eupalitin 3-O--D-galactopyranoside for
antihypertensive activity}

The eupalitin- 3-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside were evaluated for
antihypertensisve activity by non- invasive method using tail cutt
method. The results were shown in table 6 and compared with standard
drug captopril. Eupalitin was found to be show highly significant
reduction in mean arterial bllod pressure at higher dose in comparison
to standard drugs( Fig.4)

\textbf{Conclusion}

Preparation of 80\% methanolic extracts of \emph{Boerhavia diffusa} by
using Soxhlet extraction and fractionation of extracts based on the
polarity. In the present study, Simultaneously determination of
eupalitin O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in \emph{Boerhavia diffusa} leaves is
carried out by HPTLC in solvent system of An efficient HPTLC method for
method of validation and quantification of eupalitin-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside in above solvent \emph{have} been developed
firstly. Isolation and purification of eupalitin-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside was done by sedimentation method and
characterization of isolated compound was performed by Mass
spectroscopy. In-vitro Hepatoprotective Activity of isolated
eupalitin-O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside was checked in HPG2 cell line
compared with silymarin by ANNOVA technique. In vivo antihypertensive
activity of isolated eupalitin O- \selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside was done in
intoxicated rats. It is concluded that methyl-predenisolone induced
arterial hypertension was accompanied by elevation in plasma rennin
activity and administration of isolated eupalitin-O-
\selectlanguage{greek}β-\selectlanguage{english}D-galactopyranoside caused more significant drops in mean arterial
blood pressure compared with toxic group by ANNOVA technique.

\textbf{Acknowledgments:} The authors acknowledge the financial support
by National Medicinal Plant Board (NMPB), AYUSH Project No:
R\&D/P-01/2010-2011. Govt. of India. The authors extend their
appreciation to the Deanship of scientific research at King Khalid
University, Grant No: RGP2/ 191/42 Saudi Arabia for financial
assistance.

\textbf{The contribution of authors is as follows:} Conceptualization,
methodology, investigation, data curation, IUK; VA conceived the
presented project idea, writing---original draft preparation,
writing---review and editing, visualization, supervision, funding
acquisition.

\textbf{Data Availability Statement:} The data that supports the finding
of this study are also available from the corresponding author upon
request.

\textbf{Competing Interest Statement:} The authors declare no conflict
of interest.

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\includegraphics[width=0.70\columnwidth]{figures/Figure-03/Figure-03}

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