Salvia miltiorrhiza, also known as Danshen, is an herb commonly used in Chinese medicine to treat cardiovascular and other diseases. As it has recently gained more recognition, this herb is currently being investigated for its anticancer properties. In traditional Chinese medicine, the patients extracted the prescribed herbs using boiling water, then consumed the extracts as medication. However, the extraction temperature of approximately 100 'C used in this traditional method may not be the most effective condition to remove the active pharmaceutical ingredients (APIs) from the herb. Previous studies show that higher temperatures can more efficiently extract anticancer agents, such as tanshinones, from Salvia miltiorrhiza. The goal of this study is to determine the most efficient subcritical water extraction temperature that extracts the APIs from the herb. The extraction process was performed at four different temperatures: 75 'C, 100 'C, 125 'C, and 150 'C. After the high-temperature water extraction, the APIs in the water extracts were analyzed using high-performance liquid chromatography (HPLC)

Biller, Jessica, J

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1  	  EXTRACTION	  OF	  SALVIA	  MILTIORRHIZA	  USING	  SUBCRITICAL	  WATER	  by	  Jessica	  Biller	  	  A	  Senior	  Honors	  Project	  Presented	  to	  the	  	  Honors	  College	  East	  Carolina	  University	  In	  Partial	  Fulfillment	  of	  the	  Requirements	  for	  	  Graduation	  with	  Honors	  by	  Jessica	  Biller	  Greenville,	  NC	  May	  2014	  	  	  Approved	  by:	  Faculty	  Mentor	  (signature	  required):	  	   	   	   	   ______________	  	  	  	  	  	  	  	  	  __	  Dr.	  Yu	  Yang	  Professor	  of	  Chemistry	  	  2  Extraction	  of	  Salvia	  Miltiorrhiza	  Using	  Subcritical	  Water	  Introduction	  	  Traditional	  Chinese	  medicine	  (TCM)	  originated	  in	  ancient	  China	  but	  has	  evolved	  over	  the	  past	  thousands	  of	  years	  and	  is	  now	  used	  all	  over	  the	  world.	  Salvia	  miltiorrhiza,	  also	  known	  as	  Danshen	  (Figure	  1),	  is	  a	  root	  commonly	  used	  in	  Chinese	  medicine	  to	  treat	  various	  cardiovascular	  diseases.	  As	  it	  has	  gained	  more	  recognition,	  this	  herb	  is	  currently	  being	  investigated	  for	  its	  anticancer	  properties.	  In	  traditional	  Chinese	  medicine,	  the	  patients	  extract	  the	  prescribed	  herbs	  using	  boiling	  water,	  then	  consume	  the	  extracts	  as	  medication.	  However,	  the	  extraction	  temperature	  of	  approximately	  100	  °C	  used	  in	  this	  traditional	  method	  may	  not	  be	  the	  most	  effective	  condition	  to	  remove	  the	  active	  pharmaceutical	  ingredients	  (APIs)	  from	  the	  herb.	  	  	  	  	  	  	  	  The	  primary	  components	  in	  Danshen	  with	  pharmacological	  importance	  are	  the	  tanshinones.	  Tanshinone	  IIA	  (Figure	  2)	  is	  currently	  used	  in	  China	  and	  other	  neighboring	  countries	  to	  treat	  patients	  suffering	  from	  myocardial	  infarction	  (MI),	  angina	  pectoris,	  stroke,	  diabetes,	  sepsis,	  and	  other	  conditions.[2]	  Benefits	  from	  treatment	  with	  the	  herb	  are	  due	  to	  its	  improved	  microcirculatory,	  vasodilatory,	  anti-­‐coagulant,	  anti-­‐thrombotic,	  anti-­‐inflammatory,	   	  Figure	  1:	  Salvia	  miltiorrhiza[1]	  3  free	  radical	  scavenging,	  and	  mitochondria-­‐protective	  effects.[3]	  It	  is	  also	  being	  researched	  for	  its	  anti-­‐cancer	  properties;	  it	  is	  known	  to	  disrupt	  cancer	  cell	  lines	  and	  promote	  apoptosis	  of	  the	  cancerous	  cells.[4]	  Like	  Tanshinone	  IIA,	  Tanshinone	  I	  (Figure	  2)	  also	  has	  anti-­‐cancer	  properties.	  Instead	  of	  promoting	  apoptosis	  of	  the	  cancerous	  cells,	  Tanshinone	  I	  reduces	  the	  transcriptional	  activity	  of	  interleukin-­‐8,	  the	  angiogenic	  factor	  involved	  in	  cancer	  metastasis.[5]	  In	  other	  words,	  Tanshinone	  I	  prevents	  the	  cancerous	  cells	  from	  growing	  and	  spreading	  to	  different	  areas	  of	  the	  body.	  Other	  anti-­‐cancer	  compounds	  found	  in	  Salvia	  miltiorrhiza	  are	  protechualdehyde,	  caffeic	  acid,	  and	  isoferulic	  acid	  (Figure	  3).	  Our	  previous	  study	  shows	  that	  higher	  temperatures	  can	  more	  efficiently	  extract	  these	  anticancer	  agents	  from	  Salvia	  miltiorrhiza.	  	  	  	  	  	  	  	  	     	  Figure	  2:	  Active	  nonpolar	  pharmaceutical	  ingredients	  found	  in	  Salvia	  miltiorrhiza:	  Tanshinone	  IIA[6]	  and	  Tanshinone	  I[7].	              	  	  	  	  	  	  	  	  	  	   	  	  Figure	  3:	  Active	  polar	  pharmaceutical	  ingredients	  found	  in	  Salvia	  miltiorrhiza:	  protocatechualdehyde[8],	  caffeic	  acid[9],	  and	  isoferulic	  acid[10]. 4  Subcritical	  water	  extraction	  (SBWE)	  is	  an	  extraction	  technique	  that	  uses	  heated	  liquid	  water	  as	  the	  extraction	  fluid.	  Subcritical	  water	  is	  water	  that	  is	  heated	  between	  100	  °C	  and	  374	  °C	  and	  is	  applied	  with	  moderate	  pressures.	  Subcritical	  water	  is	  very	  useful	  in	  the	  separation	  field.	  At	  higher	  temperatures,	  water	  becomes	  less	  polar	  and	  is	  capable	  of	  extracting	  nonpolar	  and	  hydrophobic	  molecules	  due	  to	  its	  lowered	  dielectric	  constant.[11]	  At	  elevated	  temperatures,	  water	  can	  be	  used	  as	  a	  substitute	  solvent	  for	  methanol.	  Subcritical	  water	  extraction	  is	  a	  green	  separation	  technique	  because	  it	  is	  more	  environmentally	  friendly	  than	  using	  methanol	  or	  other	  organic	  solvents,	  which	  are	  used	  in	  other	  methods	  of	  extracting	  the	  herb.	  Subcritical	  water	  extraction	  is	  the	  best	  method	  for	  the	  extraction	  of	  this	  herb	  due	  to	  the	  elimination	  of	  organic	  solvents.	  The	  goal	  of	  this	  study	  is	  to	  determine	  the	  most	  efficient	  subcritical	  water	  extraction	  temperature	  that	  extracts	  the	  active	  pharmaceutical	  ingredients:	  protechualdehyde,	  caffeic	  acid,	  isoferulic	  acid,	  Tanshinone	  I,	  and	  Tanshinone	  IIA	  from	  Salvia	  miltiorrhiza.	  	  Experimental	  	  	   The	  Salvia	  mitiorrhiza	  root	  (Danshen)	  and	  deionized	  water	  were	  used	  in	  this	  work.	  Other	  chemicals	  used	  are	  methanol,	  dichloromethane,	  phosphoric	  acid,	  Tanshinone	  I,	  Tanshinone	  IIA,	  protocatechualdehyde,	  caffeic	  acid,	  and	  isoferulic	  acid.	  The	  SBWE	  system	  was	  homemade	  with	  an	  ISCO	  Model	  100DX	  syringe	  pump	  and	  ISCO	  Series	  D	  pump	  controller.	  The	  oven	  for	  the	  system	  was	  taken	  from	  an	  Agilent	  6890	  gas	  chromatography	  (Model:	  G1530A)	  system.	  A	  diagram	  of	  the	  system	  used	  is	  shown	  in	  Figure	  4.	  	  	  	  5  	  The	  extraction	  vessel	  was	  loaded	  with	  approximately	  1.5	  g	  of	  ground	  Danshen	  then	  filled	  with	  sand.	  A	  filter	  made	  from	  Kleenex	  was	  inserted	  in	  the	  out	  flow	  end.	  The	  extraction	  vessel	  was	  then	  connected	  inside	  the	  oven.	  Before	  starting	  the	  heating	  process,	  V2	  was	  closed	  and	  the	  extraction	  vessel	  was	  pressurized	  by	  increasing	  the	  pressure	  in	  increments	  of	  2	  atm	  until	  the	  flow	  rate	  was	  no	  longer	  negative.	  The	  pressures	  used	  were	  10,	  12,	  16,	  and	  20	  atm	  for	  75,	  100,	  125,	  and	  150	  °C,	  respectively.	  Once	  the	  vessel	  was	  pressurized,	  V1	  was	  closed	  and	  the	  heating	  began.	  The	  loaded	  extraction	  vessel	  was	  heated	  for	  60	  minutes	  with	  an	  additional	  equilibration	  time	  of	  8,	  12,	  14,	  and	  16	  minutes	  for	  75,	  100,	  125,	  and	  150	  °C,	  respectively.	  After	  heating,	  V2	  and	  V1	  were	  opened	  and	  the	  sample	  was	  collected	  at	  a	  constant	  flow	  rate	  of	  1.0	  mL/min.	  The	  sample	  collected	  in	  the	  collection	  vial	  was	  the	  water	  extract.	  The	  residue	  sample	  that	  remained	  in	  the	  vessel	  was	  removed	  and	  sonicated	  for	  approximately	  4-­‐5	  hours	  with	  approximately	  5	  mL	  of	  methanol.	  Each	  extraction	  temperature	  was	  done	  in	  triplicate.	  To	  simulate	  TCM	  practice	  of	  boiling	  the	  herb	  over	  the	  stove,	  approximately	  1.5	  g	  of	  ground	  Danshen	  was	  added	  to	  a	  100	  mL	  beaker	  with	  30	  mL	  of	  18	  MΩ	  deionized	  water.	  The	  ! Figure	  1:	  Block	  diagram	  of	  SBWE	  system	  used	  for	  extractions.	   6  beaker	  was	  covered	  with	  a	  watch	  glass	  and	  heated	  to	  a	  constant	  boil	  on	  a	  hot	  plate	  for	  60	  minutes.	  The	  herb	  residue	  and	  water	  extract	  were	  separated	  and	  treated	  with	  the	  same	  procedure	  as	  the	  other	  samples.	  Liquid-­‐liquid	  extraction	  was	  performed	  on	  each	  water	  extract.	  Dichloromethane	  (2	  mL)	  was	  added	  to	  each	  vial.	  Each	  vial	  was	  then	  vortexed	  and	  the	  nonpolar	  bottom	  layer	  was	  removed	  and	  put	  into	  a	  separate	  vial.	  Because	  dichloromethane	  is	  nonpolar,	  or	  only	  slightly	  polar,	  it	  is	  assumed	  that	  the	  tanshinones	  are	  in	  this	  layer.	  The	  remaining	  water	  layer,	  containing	  the	  polar	  compounds,	  was	  filtered	  to	  separate	  the	  remaining	  liquid	  from	  the	  solid.	  Methanol	  (1	  mL)	  was	  added	  to	  each	  filtrate.	  	   An	  internal	  standard	  solution	  was	  prepared	  and	  added	  to	  each	  solution.	  A	  paraben	  mixture,	  propyl	  4-­‐hydroxybenzoate	  (125.4	  mg),	  was	  dissolved	  by	  methanol	  in	  a	  50	  mL	  volumetric	  flask	  and	  diluted	  to	  the	  mark.	  Then,	  30	  µl	  of	  this	  internal	  standard	  was	  added	  to	  each	  of	  the	  dichloromethane	  and	  sonication	  filtrate	  vials.	  300	  µl	  of	  the	  internal	  standard	  was	  added	  to	  each	  of	  the	  water	  filtrate	  vials.	  	   High-­‐Performance	  Liquid	  Chromatography	  (HPLC,	  Shimadzu Nexera UFLC system)	  was	  used	  to	  analyze	  each	  of	  the	  samples.	  The	  mobile	  phases	  used	  were	  methanol	  and	  a	  prepared	  acidified	  water	  solution.	  The	  water	  solution	  was	  prepared	  by	  adding	  10	  mL	  of	  phosphoric	  acid	  to	  1	  L	  of	  18	  MΩ	  deionized	  water.	  The	  samples	  were	  run	  at	  25	  °C;	  the	  condition	  for	  gradient	  elution	  is	  shown	  below	  in	  Table	  1:	  	  7  Table	  1:	  HPLC	  Gradient	  Conditions	   	   	   	   	   	   	   	   	   	  	  Time	   %MeoH	  %	  H3PO4	  in	  H2O	   UV	  0.0	   2	   98	   270	  4.0	   2	   98	  	  8.0	   10	   90	  	  15.4	  	   	  254	  23.0	   30	   70	  32.0	   60	   40	  43.0	   60	   40	  	  49.0	   70	   30	  	  53.0	  	   	  270	  61.0	   80	   80	  	  62.0	   90	   10	  	  65.0	   90	   10	  	  66.0	   2	   98	  	  68.0	   2	   98	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   	  	   	  Results	  and	  Discussion	  	  	   Peak	  areas	  from	  the	  HPLC	  chromatograms	  of	  the	  different	  analytes	  were	  used	  in	  determining	  the	  concentrations	  of	  those	  analytes.	  The	  concentrations	  of	  the	  analytes	  found	  in	  the	  SBWE	  extract	  (water	  and	  dichloromethane	  layers)	  are	  shown	  in	  Table	  2.	  The	  concentrations	  of	  the	  analytes	  found	  in	  the	  herb	  residue	  are	  shown	  in	  Table	  3.	  The	  concentrations	  are	  shown	  in	  micrograms	  of	  analyte	  per	  grams	  of	  herb	  loaded	  into	  the	  column.	  Table	  2:	  Concentration	  of	  APIs	  Found	  in	  SBWE	  Extract,	  µg/g	  (%RSD)	  	  Analyte	   TCM	  Replicate	   75	  °C	   100	  °C	   125	  °C	   150	  °C	  Protocatechualdehyde	   158.5	  (80.1)	   -­‐-­‐-­‐	   231.7	  (175.3)	   867.7	  (29.0)	   876.2	  (57.2)	  Caffeic	  Acid	   -­‐-­‐-­‐	   117.1	  (340.2)	   -­‐-­‐-­‐	   9.6	  (18.2)	   11.2	  (121.8)	  Isoferulic	  Acid	   7.0	  (57.2)	   10.4	  (92.0)	   2.8	  (34.1)	   138.0	  (33.0)	   37.5	  (46.1)	  Tanshinone	  I	   0.75	  (29.8)	   0.52	  (32.2)	   0.81	  (43.2)	   2.9	  (89.5)	   3.1	  (143.8)	  Tanshinone	  IIA	   1.5	  (71.7)	   1.3	  (45.6)	   1.8	  (59.3)	   2.2	  (44.2)	   0.36	  (78.8)	  	  -­‐-­‐-­‐	  indicates	  values	  that	  could	  not	  be	  quantified	   8  	  Based	  on	  the	  concentration	  of	  each	  analyte	  obtained	  at	  each	  temperature,	  the	  extraction	  efficiency	  of	  the	  analytes	  in	  the	  SBWE	  extract	  increased	  significantly	  at	  125	  °C,	  with	  the	  exception	  of	  caffeic	  acid.	  The	  concentrations	  of	  caffeic	  acid	  show	  degradation	  with	  increasing	  temperature.	  As	  shown	  in	  Table	  2,	  the	  concentration	  of	  analytes	  found	  in	  the	  herb	  residue	  was	  greatest	  at	  125	  °C,	  with	  the	  exception	  of	  Tanshinone	  IIA.	  Although	  extraction	  at	  150	  °C	  shows	  promising	  quantities	  of	  some	  of	  the	  analytes,	  the	  degradation	  of	  the	  ingredients	  in	  the	  herb	  was	  severe	  and	  such	  a	  high	  temperature	  was	  not	  ideal	  to	  work	  with.	  The	  relative	  standard	  deviation	  values	  indicate	  significant	  error	  in	  the	  triplicate	  experiments,	  which	  could	  void	  the	  significance	  of	  the	  results.	  More	  extractions	  and	  further	  analysis	  will	  need	  to	  be	  done	  in	  the	  future	  to	  ensure	  legitimacy	  of	  the	  results.	  	  	  	  	  	  	  	  	   	  Table	  3:	  Concentration	  of	  APIs	  Found	  in	  Herb	  Residue	  after	  SBWE,	  µg/g	  (%RSD)	  	  Analyte	   TCM	  Replicate	   75	  °C	   100	  °C	   125	  °C	   150	  °C	  Protocatechualdehyde	   195.44	  (69.8)	   -­‐-­‐-­‐	   110.3	  (87.7)	   261.0	  (79.5)	   226.4	  (19.1)	  Caffeic	  Acid	   -­‐-­‐-­‐	   -­‐-­‐-­‐	   13.8	  (111.0)	   9.6	  (142.5)	   0.32	  (135.9)	  Isoferulic	  Acid	   6.9	  (23.3)	   0.34	  (155.6)	   12.7	  (105.7)	   148.7	  (138.9)	   7.1	  (49.7)	  Tanshinone	  I	   39.1	  (15.7)	   93.1	  (92.1)	   70.9	  (79.5)	   138.5	  (79.9)	   15.2	  (50.6)	  Tanshinone	  IIA	   239.1	  (21.1)	   611.1	  (76.2)	   598.2	  (49.6)	   512.3	  (61.4)	   35.1	  (30.8)	  	  -­‐-­‐-­‐	  indicates	  values	  that	  could	  not	  be	  quantified	  9  References	  	  1.	  Dharmananda,	  S.	  (n.d.).	  STATUS	  OF	  SALVIA	  IN	  ANCIENT	  CHINESE	  MEDICINE.	  Salvia	  and	  the	  	  History	  of	  Microcirculation	  Research	  in	  China.	  Retrieved	  April	  27,	  2014,	  from	  http://www.itmonline.org/arts/salvia.htm	  	  2.	  Shang,	  Q.,	  Xu,	  H.,	  &	  Huang,	  L.	  (2012).	  Tanshinone	  IIA:	  A	  Promising	  Natural	  Cardioprotective	  	  Agent.	  Evidence-­‐based	  Complementary	  and	  Alternative	  Medicine,	  ,	  1-­‐7.	  	  3.	  Gao,	  S.,	  Liu,	  Z.,	  Li,	  H.,	  Little,	  P.,	  Liu,	  P.,	  &	  Xu,	  S.	  (2012).	  Cardiovascular	  actions	  and	  therapeutic	  	  potential	 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Extraction of Salvia miltiorrhiza Using Subcritical Water

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